Influenza viruses with mutant pb2 gene segment as live attenuated vaccines

ABSTRACT

The invention provides a recombinant biologically contained influenza virus that is a PB2 knockout virus, e.g., one that is useful to generate a multivalent vaccine, and methods of making and using that virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.application Ser. No. 61/527,935, filed on Aug. 26, 2011, the disclosureof which is incorporated by reference herein.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under AI047446 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

Influenza viruses instigate annual global epidemics and sporadicpandemics. Influenza A viruses annually cause epidemics characterized bya contagious respiratory illness, mild to severe fever, and in someinstances death (Palese & Shaw, 2007). Vaccine and curative antiviralresearch that focuses on the prevention and control of this potentiallyfatal virus is warranted to avoid considerable strains on health caresystems and the global economy. Intensive research has led to thediscovery of therapeutic interventions to combat influenza infections;however, due to the virus's error-prone polymerase, the hemagglutinin(HA) and neuraminidase (NA) influenza viral proteins are subject topoint mutations, known as antigenic or genetic drift (Lin et al., 2004),that allow the virus to escape host immune responses or result in sometypes of drug resistance (Moss et al., 2010). Vaccination is one of themost effective means of preventing influenza-associated morbidity andmortality.

Currently available therapeutic and prophylactic interventions includetwo types of vaccines (i.e., inactivated and live vaccines) and twoclasses of antivirals (i.e., M2 ion channel blockers, such as amantadineand rimantadine, and neuraminidase (NA)-inhibitors, such as oseltamivirand zanamivir) (Davies et al., 1964; Hayden, 2001). Nonetheless,seasonal influenza is a contagious disease with one of the highestimpacts on public health epidemiology. Further, during the 2009-2010influenza season, a novel influenza A virus strain, the 2009 H1N1pandemic virus, emerged and spread worldwide, causing the firstinfluenza pandemic in 40 years with a considerable impact on globalhealth and economics (http://www.cdc.gov/flu/about/disease/index.htm).In the United States alone, an estimated 61 million H1N1 cases,including 274,000 hospitalizations and 12,470 deaths were reported(http://www.cde.gov/flu/about/season/index.htm).

Due to an underdeveloped or impaired immune system, young, elderly orimmuno-compromised individuals are especially susceptible to infectiousdiseases such as influenza. Several studies conducted in Japan suggestedthat high rates of influenza vaccination among school age childrenprovided protection, reduced community-wide effects, and reducedincidence and mortality of older persons from influenza infection; the2001 study reported the prevention of approximately 37,000 to 49,000deaths per year and the rise of excess mortality rates when vaccinationof schoolchildren was discontinued (Reichert et al., 2001).

Currently available inactivated influenza vaccines are associated withshort protection periods and limited efficacy, especially in youngchildren and the elderly. Due to the inability to effectively elicitcell-mediated immunity, inactivated vaccines are generally lessimmunogenic, and hence less potent, than live attenuated vaccines, whichare approved for use in a limited number of countries such as the UnitesStates. Intranasally administered live attenuated viruses are consideredsuperior to inactivated vaccines for children because they elicit robustmucosal immunity and humoral and cellular immune responses coupled withlong-lasting protective efficacy (Cox et al., 2004). However, liveattenuated vaccines are currently licensed only for individuals aged 2through 49 who lack chronic medical conditions and who are not pregnantor immunocompromised, even though licensed live attenuated influenzaviruses are considered safe and stable with respect to the underlyingrisk of the emergence of revertant viruses.

Parenterally administered inactivated vaccines are also associated withadverse or anaphylactic reactions due to virus propagation inembryonated eggs, and the propensity of egg proteins in these vaccinesto induce allergies by inducing hypersensitivity reactions insusceptible hosts. A prerequisite for successful egg-based vaccinepropagation is the selection of variants adapted to embryonated chickeneggs; a criterion that may no longer match the antigenicity ofcirculating viruses. A further complication includes the possibledepletion of chicken stocks in light of a looming zoonotic outbreak ofavian influenza pandemic, which could compromise mass vaccineproduction.

Live attenuated influenza vaccine (LAIV) was originally derived by coldadaptation of an influenza type A strain (A/Ann Arbor/6/60 H2N2) and atype B strain (B/Ann Arbor/1/66) by serial passage at sequentially lowertemperatures in specific pathogen-free primary chick kidney cells(Maassab et al., 1968). During this process, the viruses acquiredmultiple mutations in internal protein gene segments (i.e., genesencoding “internal” nonglycosylated proteins) that produced thecold-adapted (ca), temperature sensitive (ts), and attenuated (att)phenotype of the master donor viruses (MDVs). The MDVs represent theLAIV genetic backbone that is updated annually with hemagglutinin (HA)and neuraminidase (NA) genes from contemporary influenza viruses toproduce the annual trivalent formulation. Thus, each of the threeinfluenza virus strains is a 6:2 genetic reassortant virus, containingsix internal gene segments from ca, ts, and att MDVs and two genesegments (encoding the HA and NA proteins) from a wild-type influenzavirus that is selected annually by the World Health Organization and theU.S. Public Health Service.

Because multiple loci in several genes control the ca, ts, and attphenotypes of LAIV vaccine viruses, it is highly improbable that LAIVwould lose these phenotypes as a result of reversion (Kemble et al.,2003; Murphy et al., 2002). Given the error rate of 10⁻⁴ to 10⁻⁵misincorporations per nucleotide position during influenza virusreplication and the fact that at least five point mutations areresponsible for the attenuated properties of each MDV (Murphy et al.,2002; Smith et al., 1987), the probability of a LAIV vaccine virusreverting to wild-type influenza, with mutations in the five attenuatingloci, would be one in at least 10²⁰ replication cycles. In one study of135 vaccine strains recovered from young vaccinated children, noevidence of reversion was observed (Vesikari et al., 2006).

The first nasally administered LIAV was approved for use in the UnitedStates in 2003, marketed in the United States as FluMist® [InfluenzaVirus Vaccine Live, Intranasal]). Although LAIV vaccine viruses wereoriginally generated using classical reassortment, in 2008 the processtransitioned to reverse genetics technology. The genetic reassortantviruses therein are prepared using reverse genetics technology in cellculture, a technique whereby influenza viruses can be generated from DNAplasmids containing influenza genes. Three vaccine strains areformulated together to produce a trivalent LAIV vaccine in single-dosesprayers. The intranasal LAIV is currently approved in the United Statesfor use in individuals 2-49 years of age.

Live attenuated viruses are considered superior to inactivated vaccinesdue to their ability to elicit both humoral and cellular immuneresponses and hence confer advanced protection in infants and youngchildren. In particular, intranasally administered live attenuatedvaccines elicit robust mucosal immunity and cellular responses coupledwith longer lasting protective efficacies (Cox et al., 2004). Liveattenuated influenza vaccine viruses replicate primarily in the ciliatedepithelial cells of the nasopharyngeal mucosa to induce immune responses(via mucosal immunoglobulin IgA, serum IgG antibodies, and cellularimmunity), but LAIV viruses do not replicate well at the warmertemperatures found in the lower airways and lung (Murphy et al., 2002;Gruber et al., 2002). In addition, there are several advantages of acell-based (e.g., cells employed to amplify virus after virus generationusing reverse genetics) alternative over the conventional egg-basedvaccine propagation system. Cell-based vaccine studies have demonstratedsignificant advantages over egg-based vaccinology in that they are amore economically feasible, rapid, and less labor-intensive alternativewhose manufacturing capacity can be readily scaled-up in proportion todemand in the context of a pandemic. Moreover, genetic engineering ofviruses through recombinant DNA-based technologies allows theexploitation of a virus' genetic parasitism, while disarming itspathogenic power. Viruses can be rendered replication-incompetent andnon-pathogenic or manipulated to introduce and express a foreign gene ina receptive host.

SUMMARY OF THE INVENTION

The invention provides a recombinant biologically contained influenzavirus that is useful to generate a multivalent vaccine, and satisfiessafety concerns regarding pathogenicity or reversion, which virusoptionally may stably express a foreign gene and so can be effectivelytraced and have its replication easily assessed. As disclosedhereinbelow, a PB2-knock-out (PB2-KO) influenza virus was generated thatharbors a reporter gene, e.g., a fluorescent protein gene such as a GFPgene or a luciferase gene, in the coding region of its PB2 viral RNA(vRNA), where the replication of the virus was restricted to a cell linethat stably expressed the PB2 protein. The reporter gene-encoding PB2vRNA was stably incorporated into progeny viruses during replication inPB2-expressing cells, and the reporter gene was expressed in virusinfected cells with no evidence of recombination between the recombinantPB2 vRNA and the PB2 protein mRNA. Further, the HA and NA genes ofdifferent virus strains were readily accommodated by the PB2-KO virus.The PB2-KO virus was used to establish an improved assay to screenneutralizing antibodies against influenza viruses by using reporter geneexpression as an indicator of virus infection rather than observingcytopathic effect. These results indicate that the PB2-KO virus have thepotential to be a valuable tool for basic and applied influenza virologyresearch, and that may be applicable to other polymerase gene knock-outviruses, e.g., PA-KO viruses or PB1-KO viruses.

In one embodiment, the invention provides isolated infectious,biologically contained influenza virus that has a viral gene segmentthat does not comprise contiguous nucleic acid sequences correspondingto those encoding PB2 (a mutant PB2 viral gene segment), a protein whichis one of the viral RNA polymerase subunits and is essential for virusreplication. To prepare such a virus in cell culture, a cell line isemployed that expresses PB2 in trans in combination with vectors forinfluenza virus vRNA production, but not one for a wild-type PB2 viralgene segment, and in one embodiment vectors for influenza virus mRNAprotein production. The resulting virus is not competent to express PB2after infection of cells that do not express PB2 in trans or are notinfected with helper virus, which provides for a “biologicallycontained” virus. However, virions produced from cells that express PB2in trans contain PB2. Such an infectious, biologically containedinfluenza virus with a mutant PB2 viral gene segment was generated inmultiple cell lines that express PB2 in trans, such as PB2-expressing293 human embryonic kidney (293), human lung adenocarcinoma epithelial(A549), or 2,6-linked sialyltransferase-overexpressing Madin-Darbycanine kidney (MDCK) cells (AX4 cells), resulting in high virus titersof at least 10⁴, 10⁵, 10⁶, 10⁷ or 10⁸ PFU/mL, or more.

Vaccination is the primary means for prophylaxis against influenzainfection. As disclosed herein, the PB2-KO virus replicated to hightiters (>10⁸ PFU/mL) in PB2-expressing but not in normal uninfectedcells (cells that do not express PB2 in trans), accommodated HA and NAgenes of a heterologous influenza virus), stably incorporated a reportergene into progeny PB2-KO virions that was retained through sequentialpassages, and was attenuated in mice, suggesting its potential as avaccine. Its ability to express antigens and its vaccine candidacy wastested in a murine model. Significantly higher levels of IgG and IgAantibodies were induced in sera, nasal washes and broncho-alveolarlavage samples from mice immunized with only one dose of PB2-KO (GFP)virus compared to inactivated influenza vaccine. All PB2-KOvirus-treated mice survived challenge with various lethal doses of PR8.Limited replication of that virus occurs in vivo as the virus producedin cells that express PB2 in trans carries along a small amount of PB2protein into the host cell which is subsequently infected (such as ahost cell which does not itself express or comprise PB2 or comprisewild-type PB2 vRNA), thereby allowing for a limited amount (e.g., around or so) of replication to occur but without a significantinfectious process (for instance, amplification of virus titers of overabout 1000). The limited replication of the KO virus in vivo allows foran immune response that provides for a more robust immune response thaninduced by conventional inactivated influenza vaccines. It is noteworthythat the immunized mice produce antibodies against the reporter, asdetermined by an immunofluorescence assay, suggesting that PB2-KO virushas the potency of a multivalent vaccine. The PB2-KO exhibited similaror better safety and efficacy profiles when compared to controls, and soholds promise for combating influenza virus infection.

In one embodiment, the invention provides an isolated infectious,biologically contained recombinant influenza virus comprising 8 genesegments including a PA viral gene segment, a PB1 viral gene segment, amutant PB2 viral gene segment, a HA viral gene segment, a NA viral genesegment, a NP viral gene segment, a M (M1 and M2) viral gene segment,and a NS (NS1 and NS2) viral gene segment. In another embodiment, theinvention provides an isolated infectious, biologically containedrecombinant influenza virus comprising 8 gene segments including a PAviral gene segment, a PB1 viral gene segment, a mutant PB2 viral genesegment, a HA viral gene segment, a NA (NA and NB) viral gene segment, aNP viral gene segment, a M (M1 and BM2) viral gene segment, and a NS(NS1 and NS2) viral gene segment. In one embodiment, the infectious,biologically contained recombinant influenza virus has a M viral genesegment for M1 and M2. In one embodiment, the infectious, biologicallycontained recombinant influenza virus has a NA viral gene segment for NBand NA. In one embodiment, the infectious, biologically containedrecombinant influenza virus has a HEF gene segment.

In yet another embodiment, the invention provides an isolatedinfectious, biologically contained recombinant influenza viruscomprising gene segments including a PA viral gene segment, a PB1 viralgene segment, a mutant PB2 viral gene segment, a NP viral gene segment,a M viral gene segment, a NS viral gene segment (for NS1 and NS2), and aHEF viral gene segment. In one embodiment, the mutant PB2 viral genesegment includes 5′ and/or 3′ PB2 viral non-coding and codingincorporation sequences, optionally flanking a heterologous nucleotidesequence, and does not include contiguous sequences corresponding tosequences encoding a functional PB2. The PB2 open reading frame in themutant PB2 viral gene segment may be replaced with or disrupted by aheterologous nucleotide sequence, such as one that is readily detectableafter transfection or infection, e.g., a reporter gene such as a GFPgene or a luciferase gene, e.g., a Renilla luciferase gene, or a geneencoding an antigen from a pathogen. In one embodiment, the PB2 codingregion in the mutant PB2 viral gene segment may include mutations suchas insertions or deletions of one or more nucleotides or those thatresult in one or more amino acid substitutions or a stop codon, or anycombination thereof, that yields a non-functional PB2 coding sequence.In one embodiment, the heterologous nucleotide sequence is about 30 toabout 5,000, e.g., about 100 to about 4,500 or about 500 to about 4,000,nucleotides in length.

The infectious, biologically contained viruses of the invention may thusbe used as influenza vaccines to induce an immunogenic response in ahost, without the risk of symptoms associated with an infection orgenetic reversion from an attenuated to a fully infectious form. Theinfectious, biologically contained viruses of the invention may elicit abetter immune response than chemically inactivated viruses because theyare live viruses, yet because they are biologically contained, theviruses of the invention likely do not cause symptoms of the disease,which is often an issue with live attenuated vaccines. And in contrastto the use of virus-like particles (VLPs), which are non-replicative,the KO viruses of the invention contain RNA, which is an adjuvant thatenhances the host's immune response against the virus. The properties ofa PB2-KO influenza virus of the invention were surprising given that asimilar virus, a M2 deficient virus that lacks the transmembrane andcytoplasmic domains of M2 (see, Watanabe et al., J. Virol., 83:5944(2009)) grew to low titers, e.g., 10²-10³ PFU/mL, in the absence of M2supplied in trans, and so was replication-defective but not biologicallycontained.

In one embodiment, the invention provides an isolated recombinantinfectious, biologically contained influenza virus comprising 7 genesegments including a PA viral gene segment, a PB1 viral gene segment, aHA viral gene segment, a NA viral gene segment, a NP viral gene segment,a M viral gene segment, and a NS1 and NS2 viral gene segment, i.e., thevirus lacks a PB2 viral gene segment.

In one embodiment, for the 8 segment PB2-KO influenza virus having amutant PB2 viral gene segment, the mutant PB2 viral gene segment has adeletion of PB2 coding sequences, a deletion of PB2 coding sequences andan insertion of heterologous nucleotide sequences, or an insertion ofheterologous nucleotide sequences which disrupts PB2 coding sequences.That virus replicates in vitro when PB2 is supplied in trans to titersthat are substantially the same or at most 10, 100 or 1,000 fold lessthan a corresponding wild-type influenza virus, but is attenuated invivo or in vitro in the absence of PB2 supplied in trans. In oneembodiment, the deletion of PB2 coding sequences includes 1 or morecontiguous or noncontiguous nucleotides of PB2 and may include adeletion of the entire coding region, e.g., a region encoding 759 aminoacids. In one embodiment, the deletion includes at least 10%, 30%, 40%,50%, 70%, 80%, 85%, 90%, 93%, 95% and up to 99%, or a percent numericalvalue that is any integer between 10 and 99, but not all, of the PB2coding region. In one embodiment, the deletion of PB2 coding sequencesdoes not include the deletion of 5′ or 3′ coding sequences that enhanceincorporation of the resulting viral gene segment into virions, e.g.,sequences that are contiguous to 3′ or 5′ non-coding PB2 sequences,relative to a recombinant viral gene segment with only non-coding PB2incorporation sequences.

In one embodiment, the mutant PB2 gene segment may comprise an insertionof one or more nucleotides, e.g., those that result in a frame-shift, sothat functional PB2 cannot be expressed. In one embodiment, theinsertion does not include the alteration of 5′ or 3′ coding sequencesthat enhance incorporation of the gene segment into virions relative toa recombinant gene segment with only non-coding PB2 incorporationsequences.

In one embodiment, the mutant PB2 viral gene segment may comprise atleast one mutation that results in at least one amino acid substitutionrelative to a corresponding wild-type PB2 protein, e.g., a mutation thatremoves or replaces the initiator codon, or that introduces one or morestop codons into the coding region, so that functional PB2 cannot beexpressed from that viral gene segment after infection. In oneembodiment, the substitution, removal or replacement of the initiatorcodon, or introduction of the one or more stop codons in the readingframe for PB2, does not include the alteration of 5′ or 3′ codingsequences that enhance incorporation of the gene segment into virionsrelative to a recombinant gene segment with only non-coding PB2incorporation sequences.

In one embodiment of the invention, the heterologous nucleotide sequencemay encode a heterologous protein (a non-influenza viral protein such asa glycoprotein or a cytosolic, nuclear or mitochondrial specificprotein, or any antigenic protein such as an antigen from a microbialpathogen), which may confer a detectable phenotype. In one embodiment,the heterologous nucleotide sequence may be fused to truncated portionsof PB2 coding sequences, e.g., those corresponding to 5′ or 3′ PB2coding incorporation sequences, optionally forming a chimeric protein.In one embodiment, the heterologous nucleotide sequence replaces or isintroduced to sequences in the viral gene segment corresponding to thecoding region for that segment, so as not to disrupt the incorporationsequences in the coding region of the gene segment. For instance, theheterologous nucleotide sequence may be flanked by about 3 to about 400nucleotides of the 5′ and/or 3′ PB2 coding region adjacent to non-codingsequence. In one embodiment, the 3′ PB2 incorporation sequencescorrespond to nucleotides 3 to 400, nucleotides 3 to 300, nucleotides 3to 100, nucleotides 3 to 50, or any integer between 3 and 400, of theN-terminal and/or C-terminal PB2 coding region. In one embodiment, afterinfection of a host cell with the biologically contained PB2-KO virus, aheterologous protein is produced which is a fusion with the N-terminusand/or C-terminus of the remaining residues of the deleted PB2 protein.

A vector for vRNA production of the mutant PB2 gene segment isintroduced into a cell along with a vector or vectors for vRNAproduction for PA vRNA, PB1 vRNA, NP vRNA, HA vRNA, NA vRNA, M vRNA, andNS (NS1 and/or NS2) vRNA, and vectors for mRNA (protein) production forone or more of PA, PB1, PB2, and NP, or vectors for mRNA production ofup to three of PA, PB1, PB2, and NP, where the cell stably expresses theremaining viral protein(s), and optionally expresses HA, NA, M, e.g., M1and M2, NS1 and/or NS2. The vRNA for the mutant PB2 gene segment may beincorporated into virions at an efficiency that is at least 1%, 5%, 10%,or 30%, or at least 50%, that of a corresponding wild-type PB2 vRNA.

In one embodiment, the influenza virus of the invention elicits bothsystemic and mucosal immunity at the primary portal of infection. Thus,the invention provides a live, attenuated vaccine or immunogeniccomposition comprising the recombinant biologically contained virus ofthe invention, and a method of using the vaccine or immunogeniccomposition to immunize a vertebrate, e.g., an avian or a mammal, suchas a human, or induce an immune response in a vertebrate, respectively.In one embodiment, the composition or vaccine is formulated forintranasal administration. In one embodiment, the recombinantbiologically contained virus in a vaccine comprises a HA gene segmentfor influenza A virus HA, e.g., H1, H2, H3, H5, H7, or H9 HA. In oneembodiment, the HA in the recombinant biologically contained virus in avaccine is modified at the HA cleavage site. In one embodiment, thevaccine comprises at least one influenza virus strain that is differentthan the recombinant biologically contained virus of the invention, forinstance, the vaccine comprises two or three different influenzaviruses.

The invention provides a plurality of vectors to prepare an infectious,biologically contained 8 segment influenza A virus having one or morevectors which include transcription cassettes for vRNA production andtranscription cassettes for mRNA production. The transcription cassettesfor vRNA production are a transcription cassette comprising a promoterfor vRNA production, e.g., a PolI promoter, operably linked to aninfluenza virus PA DNA in an orientation for genomic viral RNAproduction linked to a transcription termination sequence that resultsin influenza virus-like vRNA termini, for instance, a PolI transcriptiontermination sequence, a transcription cassette comprising a promoter forvRNA production, e.g., a PolI promoter, operably linked to an influenzavirus PB1 DNA in an orientation for genomic viral RNA production linkedto a transcription termination sequence that results in influenzavirus-like vRNA termini, for instance, PolI transcription terminationsequence, a transcription cassette comprising a promoter for vRNAproduction, e.g., a PolI promoter, operably linked to a mutant influenzavirus PB2 DNA in an orientation for genomic viral RNA production linkedto a transcription termination sequence that results in influenzavirus-like vRNA termini, for instance, a PolI transcription terminationsequence, a transcription cassette comprising a promoter for vRNAproduction, e.g., a PolI promoter, operably linked to an influenza virusHA DNA in an orientation for genomic viral RNA production linked to atranscription termination sequence that results in influenza virus-likevRNA termini, for instance, a PolI transcription termination sequence, atranscription cassette comprising a promoter for vRNA production, e.g.,a PolI promoter, operably linked to an influenza virus NA DNA in anorientation for genomic viral RNA production linked to a transcriptiontermination sequence that results in influenza virus-like vRNA termini,for instance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus NP DNA in an orientationfor genomic viral RNA production linked to a transcription terminationsequence that results in influenza virus-like vRNA termini, forinstance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus M DNA in an orientationfor genomic viral RNA production linked to a transcription terminationsequence that results in influenza virus-like vRNA termini, forinstance, a PolI transcription termination sequence, and a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus NS (NS1 and NS2) DNA inan orientation for genomic viral RNA production linked to atranscription termination sequence that results in influenza virus-likevRNA termini, for instance, a PolI transcription termination sequence.The mutant PB2 DNA includes 5′ and 3′ incorporation sequences flanking aheterologous nucleotide sequence and does not include contiguoussequences corresponding to sequences that encode a functional PB2. Thetranscription cassettes for mRNA production are a transcription cassettecomprising a PolII promoter operably linked to a DNA coding region forinfluenza virus PA linked to a PolII transcription termination sequence,a transcription cassette comprising a PolII promoter operably linked toa DNA coding region for influenza virus PB1 linked to a PolIItranscription termination sequence, and a transcription cassettecomprising a PolII promoter operably linked to a DNA coding region forinfluenza virus NP linked to a PolII transcription termination sequence,and optionally a transcription cassette comprising a PolII promoteroperably linked to a DNA coding region for influenza virus one or moreof PB2, HA, NA, NS1, NS2, M1 and/or M2 linked to a PolII transcriptiontermination sequence. Further provided is a composition having thevectors, and a method which employs the vectors.

The invention also provides a plurality of vectors to prepare aninfectious, biologically contained 8 segment influenza B virus havingone or more vectors which include transcription cassettes for vRNAproduction and transcription cassettes for mRNA production. Thetranscription cassettes for vRNA production are a transcription cassettecomprising a promoter for vRNA production, e.g., a PolI promoter,operably linked to an influenza virus PA DNA in an orientation forgenomic viral RNA production linked to a transcription terminationsequence that results in influenza virus-like vRNA termini, forinstance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus PB1 DNA in anorientation for genomic viral RNA production linked to a transcriptiontermination sequence that results in influenza virus-like vRNA termini,for instance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to a mutant influenza virus PB2 DNA in anorientation for genomic viral RNA production linked to a transcriptiontermination sequence that results in influenza virus-like vRNA termini,for instance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus HA DNA in an orientationfor genomic viral RNA production linked to a transcription terminationsequence that results in influenza virus-like vRNA termini, forinstance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus NA and NB DNA in anorientation for genomic viral RNA production linked to a transcriptiontermination sequence that results in influenza virus-like vRNA termini,for instance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus NP DNA in an orientationfor genomic viral RNA production linked to a transcription terminationsequence that results in influenza virus-like vRNA termini, forinstance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus M DNA in an orientationfor genomic viral RNA production linked to a transcription terminationsequence that results in influenza virus-like vRNA termini, forinstance, a PolI transcription termination sequence, and a transcriptioncassette comprising a PolI promoter operably linked to an influenzavirus NS (NS1 and NS2) DNA in an orientation for genomic viral RNAproduction linked to a PolI transcription termination sequence, Themutant PB2 DNA is includes 5′ and 3′ incorporation sequences, optionallyflanking a heterologous nucleotide sequence, and does not includecontiguous sequences corresponding to sequences that encode a functionalPB2. The transcription cassettes for mRNA production are a transcriptioncassette comprising a PolII promoter operably linked to a DNA codingregion for influenza virus PA linked to a PolII transcriptiontermination sequence, a transcription cassette comprising a PolIIpromoter operably linked to a DNA coding region for influenza virus PB1linked to a PolII transcription termination sequence, and atranscription cassette comprising a PolII promoter operably linked to aDNA coding region for influenza virus NP linked to a PolII transcriptiontermination sequence, and optionally a transcription cassette comprisinga PolII promoter operably linked to a DNA coding region for influenzavirus one or more of PB2, HA, NA, NS1, NS2, M1 and/or BM2 linked to aPolII transcription termination sequence. Further provided is acomposition having the vectors and a method which employs the vectors.

In one embodiment, the promoter in a vRNA vector includes but is notlimited to a RNA polymerase I (PolI) promoter, e.g., a human RNA PolIpromoter, a RNA polymerase II (PolII) promoter, a RNA polymerase IIIpromoter, a SP6 promoter, a T7 promoter, or a T3 promoter. In oneembodiment, one or more vRNA vectors include a PolII promoter andribozyme sequences 5′ to influenza virus sequences and the same ordifferent ribozyme sequences 3′ to the influenza virus sequences. In oneembodiment, the mutant PB2 gene segment is in a vector and is operablylinked to a promoter including, but not limited to, a RNA PolI promoter,e.g., a human RNA PolI promoter, a RNA PolII promoter, a RNA polymeraseIII promoter, a SP6 promoter, a T7 promoter, or a T3 promoter. In oneembodiment, the vRNA vectors include a transcription terminationsequence including, but not limited to, a PolI transcription terminationsequence, a PolII transcription termination sequence, or a PolIIItranscription termination sequence, or one or more ribozymes.

A plurality of the vectors of the invention may be physically linked oreach vector may be present on an individual plasmid or other, e.g.,linear, nucleic acid delivery vehicle. In one embodiment, each vRNAproduction vector is on a separate plasmid. In one embodiment, each mRNAproduction vector is on a separate plasmid. In one embodiment, one ormore vectors for vRNA production are on the same plasmid (see, e.g.,U.S. published application No. 20060166321, the disclosure of which isincorporated by reference herein). In one embodiment, one or morevectors for mRNA production are on the same plasmid (see, e.g., U.S.published application No. 2006/0166321). In one embodiment, the vRNAvectors employed in the method are on one plasmid or on two or threedifferent plasmids. In one embodiment, the mRNA vectors for PA, PB1, andNP, and optionally PB2, employed in the method are on one plasmid or ontwo or three different plasmids.

Also provided is a host cell comprising a vector expressing PB2, e.g.,PB2 from PR8 or other master vaccine strain. In one embodiment, the PB2has at least 90%, 95%, 98%, 99% or 100% identity to PB2 encoded by SEQID NO:3. In one embodiment, the host cell is transduced with a viralvector, e.g., a vector which is stably maintained in the cell as anepisome or integrated into a chromosome, such as a lentiviral orretroviral vector. In one embodiment, the host cell further includes oneor more vectors which include transcription cassettes for transient vRNAproduction and transcription cassettes for transient mRNA production.The transcription cassettes for vRNA production are a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus PA DNA in an orientationfor genomic viral RNA production linked to a transcription terminationsequence that results in influenza virus-like vRNA termini, forinstance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter operably linked to an influenza virus PB1 DNA in an orientationfor genomic viral RNA production linked to a transcription terminationsequence that results in influenza virus-like vRNA termini, forinstance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to a mutant influenza virus PB2 DNA in anorientation for genomic viral RNA production linked to a transcriptiontermination sequence that results in influenza virus-like vRNA termini,for instance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus HA DNA in an orientationfor genomic viral RNA production linked to a transcription terminationsequence that results in influenza virus-like vRNA termini, forinstance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus NA DNA in an orientationfor genomic viral RNA production linked to a transcription terminationsequence that results in influenza virus-like vRNA termini, forinstance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus NP DNA in an orientationfor genomic viral RNA production linked to a transcription terminationsequence that results in influenza virus-like vRNA termini, forinstance, a PolI transcription termination sequence, a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus M DNA in an orientationfor genomic viral RNA production linked to a transcription terminationsequence that results in influenza virus-like vRNA termini, forinstance, a PolI transcription termination sequence, and a transcriptioncassette comprising a promoter for vRNA production, e.g., a PolIpromoter, operably linked to an influenza virus NS (NS1 and NS2) DNA inan orientation for genomic viral RNA production linked to atranscription termination sequence that results in influenza virus-likevRNA termini, for instance, a PolI transcription termination sequence.The mutant PB2 DNA includes 5′ and 3′ incorporation sequences,optionally flanking a heterologous nucleotide sequence, and does notinclude contiguous sequences corresponding to sequences that encode afunctional PB2. The transcription cassettes for mRNA production are atranscription cassette comprising a PolII promoter operably linked to aDNA coding region for influenza virus PA linked to a PolII transcriptiontermination sequence, a transcription cassette comprising a PolIIpromoter operably linked to a DNA coding region for influenza virus PB1linked to a PolII transcription termination sequence, and atranscription cassette comprising a PolII promoter operably linked to aDNA coding region for influenza virus NP linked to a PolII transcriptiontermination sequence. The host cell does not include sequencescorresponding to PB2 coding sequences for vRNA production of a wild-typePB2 viral gene segment.

The invention also provides a method to prepare influenza virus, e.g.,using a host cell of the invention. The method comprises contacting acell with a plurality of the vectors of the invention, e.g.,sequentially or simultaneously, in an amount effective to yieldinfectious influenza virus. The invention also includes isolating virusfrom a cell contacted with the plurality of vectors. Thus, the inventionfurther provides isolated virus, as well as a host cell contacted withvirus of the invention. In another embodiment, the invention includescontacting the cell with one or more vectors, either vRNA or proteinproduction vectors, prior to other vectors, either vRNA or proteinproduction vectors.

In one embodiment, the invention provides a method of preparing arecombinant influenza virus comprising a mutant PB2 viral gene segment.The method comprises contacting a host cell with a plurality ofinfluenza vectors, including a vector comprising the mutant PB2 genesegment sequence, so as to yield recombinant virus. For example, thehost cell is contacted with vectors for vRNA production including avector comprising a promoter for vRNA production operably linked to aninfluenza virus PA DNA linked to a transcription termination sequence, avector comprising a promoter for vRNA production operably linked to aninfluenza virus PB1 DNA linked to a transcription termination sequence,a vector comprising a promoter for vRNA production operably linked to amutant influenza virus PB2 DNA linked to a transcription terminationsequence, a vector comprising a promoter for vRNA production operablylinked to an influenza virus HA DNA linked to a transcriptiontermination sequence, a vector comprising a promoter for vRNA productionoperably linked to an influenza virus NP DNA linked to a transcriptiontermination sequence, a vector comprising a promoter for vRNA productionoperably linked to an influenza virus NA DNA linked to a transcriptiontermination sequence, a vector comprising a promoter for vRNA productionoperably linked to an influenza virus M DNA linked to a transcriptiontermination sequence, and a vector comprising a promoter for vRNAproduction operably linked to an influenza virus NS (NS1 and NS2) DNAlinked to a transcription termination sequence, wherein the mutant PB2DNA is in an orientation for genomic vRNA production and includes 5′ and3′ incorporation sequences, optionally flanking a heterologousnucleotide sequence, and does not include contiguous sequencescorresponding to those for a functional PB2, and vectors for mRNAproduction including a vector comprising a promoter operably linked to aDNA segment encoding influenza virus PA, a vector comprising a promoteroperably linked to a DNA segment encoding influenza virus PB1, a vectorcomprising a promoter operably linked to a DNA segment encodinginfluenza virus NP, wherein the cell is not contacted with sequencescorresponding to PB2 coding sequences for vRNA production. Optionally,the host cell is contacted with a vector comprising a promoter operablylinked to a DNA segment encoding influenza virus HA, a vector comprisinga promoter operably linked to a DNA segment encoding influenza virus NA,a vector comprising a promoter operably linked to a DNA segment encodinginfluenza virus M1, a vector comprising a promoter operably linked to aDNA segment encoding a M2 protein, e.g., a mutant M2 protein, and avector comprising a promoter operably linked to a DNA segment encodinginfluenza virus NS1 and/or NS2. In one embodiment, separate vectors forM1 and M2 mRNA, and/or for NS1 and NS2 mRNA are provided and employed.

In one embodiment of a method of preparing a recombinant biologicallycontained influenza virus of the invention, each transcription cassetteis on a plasmid vector. In one embodiment of a method of preparing abiologically contained influenza virus of the invention, one or moretranscription cassettes are on one or more plasmid vectors, e.g., oneplasmid vector has transcription cassettes for vRNA production of PA,PB1, HA, NP, NA, M1, NS1 and/or NS2, and the mutant PB2 cDNAs. In oneembodiment of a method of preparing a biologically contained influenzavirus of the invention, one plasmid vector has one of the transcriptioncassette for mRNA production and another plasmid vector has the othertranscription cassettes for mRNA production. In one embodiment of amethod of preparing a biologically contained influenza virus of theinvention, three plasmid vectors for mRNA production are employed, eachwith one of the transcription cassettes for mRNA production. In oneembodiment of a method of preparing a biologically contained influenzavirus of the invention, one plasmid vector has six of the transcriptioncassettes for vRNA production and another plasmid vector has the othertranscription cassette for vRNA production, e.g., one plasmid vector hasone of the transcription cassettes for mRNA production and anotherplasmid vector has the other transcription cassettes for mRNAproduction. In one embodiment of a method of preparing a biologicallycontained influenza virus of the invention, three plasmid vectors formRNA production are employed. In one embodiment of a method of preparinga biologically contained influenza virus of the invention, one plasmidhas the three transcription cassettes for mRNA production. In oneembodiment of a method of preparing a biologically contained influenzavirus of the invention, the HA cDNA encodes an avirulent cleavage site.In one embodiment of a method of preparing a biologically containedinfluenza virus of the invention, the HA and NA are from the same virusisolate. In one embodiment of a method of preparing a biologicallycontained influenza virus of the invention, the HA is a type B HA.

The promoter or transcription termination sequence in a vRNA or virusprotein expression vector may be the same or different relative to thepromoter or any other vector. In one embodiment, the vector or plasmidwhich expresses influenza vRNA comprises a promoter suitable forexpression in at least one particular host cell, e.g., avian ormammalian host cells such as canine, feline, equine, bovine, ovine, orprimate cells including human cells, or for expression in more than onehost. In one embodiment, the PolI promoter for each PolI containingvector is the same. In one embodiment, the PolI promoter is a human PolIpromoter. In one embodiment, the PolII promoter for each PolIIcontaining vector is the same. In one embodiment, the PolII promoter fortwo or more, but not all, of the PolII containing vectors, is the same.In one embodiment, the PolII promoter for each PolII containing vectoris different.

In another embodiment, the method includes contacting a host cell with avector having a PolII promoter linked to a PolI transcriptiontermination sequence linked to an influenza virus PA DNA linked to aPolI promoter linked to a PolII transcription termination sequence (abidirectional cassette), a vector having a PolII promoter linked to aPolII transcription termination sequence linked to an influenza virusPB1 DNA linked to a PolI promoter linked to a PolII transcriptiontermination sequence, a vector having a PolII promoter linked to a PolItranscription termination sequence linked to a mutant influenza virusPB2 DNA linked to a PolI promoter linked to a PolII transcriptionterminator sequence, a vector having a PolII promoter linked to a PolItranscription termination sequence linked to an influenza virus HA DNAlinked to a PolI promoter linked to a PolII transcription terminationsequence, a vector having a PolII promoter linked to a PolItranscription termination sequence linked to an influenza virus NP DNAlinked to a PolI promoter linked to a PolII transcription terminationsequence, a vector having a PolII promoter linked to a PolItranscription termination sequence linked to an influenza virus NA DNAlinked to a PolI promoter linked to a PolII transcription terminationsequence, a vector having a PolII promoter linked to a PolItranscription termination sequence linked to an influenza virus M DNAlinked to a PolI promoter linked to PolII transcription terminationsequence, and a vector having a PolII promoter linked to a PolItranscription termination sequence linked to an influenza virus NS1and/or NS2 DNA linked to a PolI promoter linked to PolII transcriptiontermination sequence. The host cell comprises PB2 DNA expressing a PB2protein, e.g., from a chicken beta-actin promoter. No sources of vRNAfor wild-type PB2 are present so that replication-incompetent virus isprovided.

In one embodiment, the promoter for vRNA production in a bidirectionalcassette includes but is not limited to a RNA polymerase I (PolI)promoter, e.g., a human RNA PolI promoter, a RNA polymerase II (PolII)promoter, a RNA polymerase III promoter, a SP6 promoter, a T7 promoter,or a T3 promoter. In one embodiment, one or more vRNA vectors include aPolII promoter and ribozyme sequences 5′ to influenza virus sequencesand the same or different ribozyme sequences 3′ to the influenza virussequences. In one embodiment, the mutant PB2 gene segment is in a vectorand is operably linked to a promoter including, but not limited to, aRNA PolI promoter, e.g., a human RNA PolI promoter, a RNA PolIIpromoter, a RNA polymerase III promoter, a SP6 promoter, a T7 promoter,or a T3 promoter. In one embodiment, the vRNA vectors include atranscription termination sequence including, but not limited to, a PolItranscription termination sequence, a PolII transcription terminationsequence, or a PolIII transcription termination sequence, or one or moreribozymes. Ribozymes within the scope of the invention include, but arenot limited to, tetrahymena ribozymes, RNase P, hammerhead ribozymes,hairpin ribozymes, hepatitis ribozyme, as well as synthetic ribozymes.In one embodiment, at least one vector for vRNA comprises a RNApolymerase II promoter linked to a ribozyme sequence linked to viralcoding sequences linked to another ribozyme sequences, optionally linkedto a RNA polymerase II transcription termination sequence. In oneembodiment, at least 2, e.g., 3, 4, 5, 6, 7 or 8, vectors for vRNAproduction comprise a RNA polymerase II promoter, a first ribozymesequence, which is 5′ to a sequence corresponding to viral sequencesincluding viral coding sequences, which is 5′ to a second ribozymesequence, which is 5′ to a transcription termination sequence. Each RNApolymerase II promoter in each vRNA vector may be the same or differentas the RNA polymerase II promoter in any other vRNA vector. Similarly,each ribozyme sequence in each vRNA vector may be the same or differentas the ribozyme sequences in any other vRNA vector. In one embodiment,the ribozyme sequences in a single vector are not the same.

The plurality of vectors, compositions and host cells of the inventionmay also include another vector for vRNA production or proteinproduction that includes heterologous sequences, e.g., for a therapeuticor prophylactic gene of interest e.g., an immunogen for a cancerassociated antigen or for a pathogen such as a bacteria, a noninfluenzavirus, fungus, or other pathogen. For example, the vector or plasmidcomprising the gene or cDNA of interest may substitute for a vector orplasmid for an influenza viral gene or may be in addition to vectors orplasmids for all influenza viral genes. Thus, another embodiment of theinvention comprises a composition or plurality of vectors as describedabove in which one of the vectors is replaced with, or furthercomprises, 5′ influenza virus sequences optionally including 5′influenza virus coding sequences or a portion thereof, linked to adesired nucleic acid sequence, e.g., a desired cDNA, linked to 3′influenza virus sequences optionally including 3′ influenza virus codingsequences or a portion thereof. In one embodiment, the desired nucleicacid sequence such as a cDNA is in an antisense (antigenomic)orientation. The introduction of such a vector in conjunction with theother vectors described above to a host cell permissive for influenzavirus replication results in recombinant virus comprising vRNAcorresponding to the heterologous sequences of the vector.

In one embodiment, the recombinant virus of the invention includes oneor more genes from influenza A virus. In another embodiment, therecombinant virus of the invention may include one or more genes frominfluenza B virus, e.g., an influenza B HA gene. In yet anotherembodiment, the recombinant virus of the invention may include one ormore genes from influenza C virus. The DNA for vRNA production of NA maybe from any NA, e.g., any of N1-N9, a chimeric NA sequence or anynon-native NA sequence, and the DNA for vRNA production of HA may befrom any HA, e.g., H1-H16, a chimeric HA sequence or any non-native HAsequence. In one embodiment, the DNAs for vRNA production may be for aninfluenza B or C virus. In one embodiment, other attenuating mutationsmay be introduced to the vectors, e.g., a mutation in a HA cleavage sitethat results in a site that is not cleaved. The DNAs for vRNA productionof NA and HA may be from different strains or isolates relative to thosefor the (6:1:1 reassortants) or from the same strain or isolate (6:2reassortants), the NA may be from the same strain or isolate as that forthe internal genes (7:1 reassortant), or one of the internal genes, NAand HA may be from the same strain or isolate (5:3 reassortant).

Viruses that may provide the internal genes for reassortants within thescope of the invention include viruses that have high titers in Verocells, e.g., titers of at least about 10⁵ PFU/mL, e.g., at least 10⁶PFU/mL, 10⁷ PFU/mL or 10⁸ PFU/mL; high titers in embryonated eggs, e.g.,titers of at least about 10⁷ EID₅₀/mL, e.g., at least 10⁸ EID₅₀/mL, 10⁹EID₅₀/mL or 10¹⁰ EID₅₀/mL; high titers in MDCK, e.g., AX5, cells, e.g.,titers of at least about 10⁷ PFU/mL, e.g., at least 10⁸ PFU/mL, or hightiters in two of more of those host cells. In one embodiment, the DNAsfor vRNA production of PB1 vRNA, mutant PB2 vRNA, PA vRNA, NP vRNA, MvRNA (for M1 and/or M2 or M1 and/or BM2), and/or NS vRNA (for NS1 and/orNS2), may have sequences from an influenza virus that replicates to hightiters in cultured mammalian cells such as AX4 cells, Vero cells orPER.C6® cells and also optionally embryonated eggs, and/or from avaccine virus, e.g., one that does not cause significant disease inhumans.

For example, reassortants with internal genes from other PR8 isolates orvaccine viruses may be employed in recombinant reassortant viruses ofthe invention. In particular, 5:1:2 reassortants having PR8(UW) PB1,PB2, PA, NP, and M (“5”) and PR8(Cam) NS (“1”); 6:1:1 reassortantshaving PR8(UW) NA, PB1, PB2, PA, NP, and M (“6”) and PR8(Cam) NS (“1”)gene segments; and 7:1 reassortants having PR8(UW) PB1, PB2, PA, NP, M,NA, and NS (“7”) gene segments may be employed.

In one embodiment, the DNAs for the internal genes for PB1, PB2, PA, NP,M, and NS encode proteins with substantially the same activity as acorresponding polypeptide encoded by one of SEQ ID NOs:1-6 or 10-15. Asused herein, “substantially the same activity” includes an activity thatis about 0.1%, 1%, 10%, 30%, 50%, 90%, e.g., up to 100% or more, ordetectable protein level that is about 80%, 90% or more, the activity orprotein level, respectively, of the corresponding full-lengthpolypeptide. In one embodiment, the nucleic acid a sequence encoding apolypeptide which is substantially the same as, e.g., having at least80%, e.g., 90%, 92%, 95%, 97% or 99%, including any integer between 80and 99, contiguous amino acid sequence identity to, a polypeptideencoded by one of SEQ ID NOs:1-6 or 10-15. In one embodiment, theisolated and/or purified nucleic acid molecule comprises a nucleotidesequence which is substantially the same as, e.g., having at least 50%,e.g., 60%, 70%, 80% or 90%, including any integer between 50 and 100, ormore contiguous nucleic acid sequence identity to one of SEQ ID NOs:1-6or 33-38 and, in one embodiment, also encodes a polypeptide having atleast 80%, e.g., 90%, 92%, 95%, 97% or 99%, including any integerbetween 80 and 99, contiguous amino acid sequence identity to apolypeptide encoded by one of SEQ ID NOs:1-6 or 10-15. In oneembodiment, the influenza virus polypeptide has one or more, forinstance, 2, 5, 10, 15, 20 or more, conservative amino acidssubstitutions, e.g., conservative substitutions of up to 10% or 20% ofthe residues, relative to a polypeptide encoded by one of SEQ ID NOs:1-6or 10-15. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine and tryptophan; a group of amino acids having basic side chainsis lysine, arginine and histidine; and a group of amino acids havingsulfur-containing side chain is cysteine and methionine. In oneembodiment, conservative amino acid substitution groups are:valine-leucine-isoleucine; phenylalanine-tyrosine; lysine-arginine;alanine-valine; glutamic-aspartic; and asparagine-glutamine. In oneembodiment, the influenza virus polypeptide has one or more, forinstance, 2, 3 or 4, nonconservative amino acid substitutions, relativeto a polypeptide encoded by one of SEQ ID NOs:1-6 or 10-15.

The methods of producing virus described herein, which do not requirehelper virus infection, are useful in viral mutagenesis studies, and inthe production of vaccines (e.g., for AIDS, influenza, hepatitis B,hepatitis C, rhinovirus, filoviruses, malaria, herpes, and foot andmouth disease) and gene therapy vectors (e.g., for cancer, AIDS,adenosine deaminase, muscular dystrophy, ornithine transcarbamylasedeficiency and central nervous system tumors). Thus, a virus for use inmedical therapy (e.g., for a vaccine or gene therapy) is provided.

The methods include administering to a host organism, e.g., a mammal, aneffective amount of the influenza virus of the invention, e.g., a liveor inactivated virus preparation, optionally in combination with anadjuvant and/or a carrier, e.g., in an amount effective to prevent orameliorate infection of an animal such as a mammal by that virus or anantigenically closely related virus. In one embodiment, the virus isadministered intramuscularly while in another embodiment, the virus isadministered intranasally. In some dosing protocols, all doses may beadministered intramuscularly or intranasally, while in others acombination of intramuscular and intranasal administration is employed.In one embodiment, two to three doses are administered. The vaccine maybe multivalent as a result of the heterologous nucleotide sequenceintroduced into a viral gene segment in the influenza virus of theinvention. The vaccine may further contain other isolates of influenzavirus including recombinant influenza virus, other pathogen(s),additional biological agents or microbial components, e.g., to form amultivalent vaccine. In one embodiment, intranasal vaccination, forinstance containing with inactivated influenza virus, and a mucosaladjuvant may induce virus-specific IgA and neutralizing antibody in thenasopharynx as well as serum IgG.

The influenza virus of the invention may employed with otheranti-virals, e.g., amantadine, rimantadine, and/or neuraminidaseinhibitors, e.g., may be administered separately in conjunction withthose anti-virals, for instance, administered before, during and/orafter.

In one embodiment, the influenza viruses of the invention may be vaccinevectors for influenza virus and for at least one other pathogen, such asa viral or bacterial pathogen, or for a pathogen other than influenzavirus, pathogens including but not limited to, lentiviruses such as HIV,hepatitis B virus, hepatitis C virus, herpes viruses such as CMV or HSV,Foot and Mouth Disease Virus, Measles virus, Rubella virus, Mumps virus,human Rhinovirus, Parainfluenza viruses, such as respiratory syncytialvirus and human parainfluenza virus type 1, Coronavirus, Nipah virus,Hantavirus, Japanese encephalitis virus, Rotavirus, Dengue virus, WestNile virus, Streptococcus pneumoniae, Mycobacterium tuberculosis,Bordetella pertussis, or Haemophilus influenza. For example, thebiologically contained influenza virus of the invention may includesequences for H protein of Measles virus, viral envelope protein E1 ofRubella virus, HN protein of Mumps virus, RV capsid protein VP1 of humanRhinovirus, G protein of Respiratory syncytial virus, S protein ofCoronavirus, G or F protein of Nipah virus, G protein of Hantavirus, Eprotein of Japanese encephalitis virus, VP6 of Rotavirus, E protein ofDengue virus, E protein of West Nile virus, PspA of Streptococcuspneumonia, HSP65 from Mycobacterium tuberculosis, IRP1-3 of Bordetellapertussis, or the heme utilization protein, protective surface antigenD15, heme binding protein A, or outer membrane protein P1, P2, P5 or P6of Haemophilus influenza.

Further provided is a method to detect neutralizing antibodies for aselected influenza virus strain in a physiological sample of avertebrate. The method includes contacting the sample, a recombinantvirus of the invention which expresses HA and/or NA of the selectedstrain, and cells susceptible to influenza virus infection. The presenceor amount of the reporter or the antigen in the cells is detected,wherein the absence of the reporter or antigen or a reduced amount ofthe reporter or antigen in the sample relative to a control sample, isindicative of a vertebrate that has been infected with the influenzavirus strain.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Schematic diagram of mutant PB2 vRNAs and their efficiencies invirion formation and virion incorporation. The numbers of VLPs and thevirion incorporation efficiencies of mutant PB2 vRNAs were determined byusing the numbers of WSN HA- and GFP-expressing cells as a denominator.All mutants are shown in the negative-sense orientation. Each mutantcontains the GFP reading frame (green bar); 27 and 34 nucleotides of the3′ and 5′ noncoding regions, respectively (gray bars); and codingregions of various lengths (black bars). The dotted lines representdeleted sequences of the PB2 coding region. PB2(−) indicates theomission of this vRNA (i.e., VLPs were generated using only seven vRNAsegments).

FIG. 2. Exemplary vaccine virus internal gene sequences (SEQ ID NOs:1-8and 10-15).

FIG. 3. Characterization of PR8/PB2-GFP virus. A) Schematic diagram ofwild-type PB2 and PB2(120)GFP(120) vRNAs. PB2(120)GFP(120) vRNApossesses the 3′ noncoding region, 120 nucleotides of the codingsequence of PB2 vRNA, the GFP gene, and 120 nucleotides of the 3′ codingand the 5′ noncoding regions of PB2 vRNA. The noncoding region andcoding regions of PB2 vRNA are represented by gray and red bars,respectively. The GFP gene is represented by the green bar. B) PB2 geneexpression in AX4/PB2 cells (AX4 cells are derived from MDCK cells). RNAwas extracted from both wild-type AX4 and AX4/PB2 cells. RT-PCR wasperformed by using an oligo(dT) primer followed by cDNA synthesis andPCR with PB2—(upper panel) or canine beta-actin—(lower panel) specificprimers. C) PB2 protein expression in AX4/PB2 cells. Cells were reactedwith an anti-PB2 antibody 18/1 (Hatta et al., 2000) (left panels) andHoechst 33342 (right panels). Scale bar, 50 μm. D) Growth kinetics ofPR8/PB2-GFP monitored over 72 hours. Wild-type AX4 (upper panel) andAX4/PB2 (lower panel) cells were infected with wild-type PR8 (red) orPR8/PB2-GFP (green) viruses at an MOI of 0.001. Supernatants collectedat the indicated time points were assayed for infectious virus in plaqueassays in AX4/PB2 cells.

FIG. 4. Accommodation of various HA genes in PB2-KO virus. HA expressionin PB2-KO virus-infected cells. AX4/PB2 cells were infected withPR8/PB2-GFP, WSN/PB2-GFP, CA04/PB2-GFP, or VN1203/PB2-GFP. At 16 hourspost-infection, the cells were stained with monoclonal antibodiesspecific for WSN, CA04, or VN1203 HA protein. The expression of HA andGFP were examined by using fluorescence microscopy.

FIG. 5. Accommodation of various reporter genes in PB2-456 KO virus. A)Luciferase activity in PB2-KO virus-infected cells. Wild-type AX4 andAX4/PB2 cells were infected with PR8/PB2-Fluc (upper graph) andPR8/PB2-Rluc (lower graph) at the indicated MOIs. At 8 hourspost-infection, Fluc and Rluc activities in cells were measured by usinga dual-luciferase reporter assay system. Results from virus-infectedcells were compared with those from uninfected cells (indicated by ‘0’)and P values were calculated by using the Student's t test. Asterisk,P<0.05. RLU, relative light unit. B) GFP intensity in PB2-KOvirus-infected cells. AX4/PB2 cells were infected with PR8/PB2-GFP atthe indicated MOI. At 8 hours post-infection, GFP intensity was measuredwith the Infinite M1000 microplate reader. Results from virus-infectedcells were compared with those from uninfected cells (indicated by ‘0’)and P values were calculated by using the Student's t test. Asterisk,P<0.05. RFU, relative fluorescent unit.

FIG. 6. PB2-KO virus-based microneutralization assay. AX4/PB2 cells wereinfected with 100 PFU of CA04/PB2-Rluc that were pre-mixed with seriallydiluted ferret sera in triplicate wells. Rluc activity in cells wasmeasured by using a Renilla luciferase assay system at 24 hourspost-infection. Results from virus-infected cells were compared withthose from cells that were infected with serum-untreated virus(indicated by ‘Serum (−)’). P values were calculated by using theStudent's t test. Asterisk, P<0.05. RLU, relative light unit.

FIG. 7. Body weight change after challenge in mice. Mice immunized withthe indicated agents once (A), twice (B), or three times (C) werechallenged with 0.5 (i) or 5 (ii) MLD₅₀ of PR8 virus. Values areexpressed as mean changes in body weight ±SD (n=3).

FIG. 8. Virus-specific antibody responses in immunized mice. PurifiedPR8 virus was used as an antigen to analyze IgG and IgA antibody titersin the sera, nasal washes, and BAL fluids (top, middle, and bottom,respectively) of mice mock immunized with medium or immunized with theformalin-inactivated virus or with the PB2-KO virus. Sera (top panels)were obtained at different time points, i.e., prevaccination (bars A),before the second vaccination (bars B), before the third vaccination(bars C), and before challenge (bars D). Nasal washes and BAL fluids(middle and bottom panels, respectively) were obtained 1 day beforechallenge from mice given 1 vaccination (bars 1), 2 vaccinations (bars2), or 3 vaccinations (bars 3). Values are expressed as the meanabsorbance ±standard deviation (SD) (n=3). Statistical significancebetween samples obtained from mice immunized with inactivated virus andPB2-KO virus is indicated.

FIG. 9. Virus titers in the lungs and nasal turbinates (NT) of immunizedmice after challenge. The numbers on the x axis indicate the number ofvaccinations. Three BALB/c mice per group were intranasally infectedwith the indicated doses of PR8 virus (50 μL per mouse) and sacrificedon days 3 and 6 postinfection for virus titration. Bars indicate thevirus titer in each organ of each mouse. The absence of bars indicatesthat virus titers were below the detection limit of 5 PFU/mL/organ.

FIG. 10. Detection of antibodies against GFP in the sera of miceimmunized with the PB2-KO virus. Confluent 293 cells that transientlyexpress GFP were treated with sera (1/20 dilution) obtained from miceinoculated with medium (A), the formalin-inactivated virus (B), or thePB2-KO virus (C) or were treated with a commercial anti-GFP antibody(D). DNA (first column) was stained with Hoechst 33342. GFP (secondcolumn) represents cells transfected with a plasmid for the expressionof GFP. GFP antibody (third column) represents the presence of the GFPantibody in the samples. These three images were merged (fourth column).Scale bars, 20 μm.

FIG. 11. Detection of heterologous antigen expression after infection ofcells with PB2-KO virus having sequences for pspA of pneumococcus (S.pneumoniae). Anti-influenza virus antibodies and anti-PspA antibodieswere used to detect expression of influenza virus and PspA proteins incells with PB2-KO-GFP or PB2-KO-PspA.

FIG. 12. Growth kinetics of PB2-KO-PspA in cells that do not express PB2and cells that stably express PB2.

FIG. 13. Influenza antigen specific IgG and IgA in serum, BAL and nasalwashes from mice immunized three times with PB2-KO-PspA or PB2-KO-GFP.

FIG. 14. PspA specific IgG in serum from mice immunized three times withPB2-KO-PspA.

FIG. 15. PspA specific IgG and IgA in BAL and nasal washes from miceimmunized three times with PB2-KO-PspA.

FIG. 16. Survival post-challenge of mice immunized three times withPB2-KO-PspA and challenged with 10 LD₅₀ or 100 LD₅₀ of influenza virus.

FIG. 17. Virus replication in the respiratory tract at day 3post-challenge in mice immunized three times with PB2-KO-PspA andchallenged with 10 LD₅₀ or 100 LD₅₀ of influenza virus.

FIG. 18. Number of pneumococci in nasal washes from mice immunized threetimes with PB2-KO-PspA or PB2-KO-GFP and challenged with 10² CFU S.pneumoniae (EF3030)/mouse.

FIG. 19. Survival post-challenge of mice immunized three times withPB2-KO-PspA and challenged with 2×10⁷ CFU pneumococcus WU2 (a lethalstrain)/mouse.

DETAILED DESCRIPTION Definitions

As used herein, an “infectious, biologically contained” virus means thatthe virus is incapable of producing progeny in normal cells or incapableof significant replication in vitro or in vivo, e.g., titers of lessthan about 10² to 10³ PFU/mL, in the absence of helper virus or a viralprotein stably supplied in trans.

A used herein, “replication-deficient” virus means that the virus canreplicate to a limited extent in vitro or in vivo, e.g., titers of atleast about 10² to 10³ PFU/mL, in the absence of helper virus or a viralprotein supplied in trans.

As used herein, the term “isolated” refers to in vitro preparationand/or isolation of a nucleic acid molecule, e.g., vector or plasmid,peptide or polypeptide (protein), or virus of the invention, so that itis not associated with in vivo substances, or is substantially purifiedfrom in vitro substances. An isolated virus preparation is generallyobtained by in vitro culture and propagation, and/or via passage ineggs, and is substantially free from other infectious agents.

As used herein, “substantially purified” means the object species is thepredominant species, e.g., on a molar basis it is more abundant than anyother individual species in a composition, and preferably is at leastabout 80% of the species present, and optionally 90% or greater, e.g.,95%, 98%, 99% or more, of the species present in the composition.

As used herein, “substantially free” means below the level of detectionfor a particular infectious agent using standard detection methods forthat agent.

A “recombinant” virus is one which has been manipulated in vitro, e.g.,using recombinant DNA techniques, to introduce changes to the viralgenome. Reassortant viruses can be prepared by recombinant ornonrecombinant techniques.

As used herein, the term “recombinant nucleic acid” or “recombinant DNAsequence or segment” refers to a nucleic acid, e.g., to DNA, that hasbeen derived or isolated from a source, that may be subsequentlychemically altered in vitro so that its sequence is not naturallyoccurring or corresponds to naturally occurring sequences that are notpositioned as they would be positioned in the native genome. An exampleof DNA “derived” from a source, would be a DNA sequence that isidentified as a useful fragment, and which is then chemicallysynthesized in essentially pure form. An example of such DNA “isolated”from a source would be a useful DNA sequence that is excised or removedfrom said source by chemical means, e.g., by the use of restrictionendonucleases, so that it can be further manipulated, e.g., amplified,for use in the invention, by the methodology of genetic engineering.

As used herein, a “heterologous” nucleotide sequence is from a sourceother than a parent influenza virus, e.g., a reporter gene or a genefrom another virus or organism, e.g., a bacterium, or is from aninfluenza virus source but is in a context that does not mimic a nativeinfluenza virus genome, e.g., it is a subset of a full length influenzavirus gene segment and is in a non-native context, e.g., fused totruncated PB2 coding sequences.

As used herein, a “heterologous” influenza virus gene or gene segment isfrom an influenza virus source that is different than a majority of theother influenza viral genes or gene segments in a recombinant, e.g.,reassortant, influenza virus.

The terms “isolated polypeptide”, “isolated peptide” or “isolatedprotein” include a polypeptide, peptide or protein encoded by cDNA orrecombinant RNA including one of synthetic origin, or some combinationthereof.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule expressed from a recombinant DNAmolecule. In contrast, the term “native protein” is used herein toindicate a protein isolated from a naturally occurring (i.e., anonrecombinant) source. Molecular biological techniques may be used toproduce a recombinant form of a protein with identical properties ascompared to the native form of the protein.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Alignments using these programs can be performed using the defaultparameters. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). The algorithm may involve firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold. These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when the cumulative alignmentscore falls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below due to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached.

In addition to calculating percent sequence identity, the BLASTalgorithm may also perform a statistical analysis of the similaritybetween two sequences. One measure of similarity provided by the BLASTalgorithm may be the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acidsequence to the reference nucleic acid sequence is less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

The BLASTN program (for nucleotide sequences) may use as defaults awordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5,N=−4, and a comparison of both strands. For amino acid sequences, theBLASTP program may use as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix. Seehttp://www.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Influenza Virus

The life cycle of viruses generally involves attachment to cell surfacereceptors, entry into the cell and uncoating of the viral nucleic acid,followed by replication of the viral genes inside the cell. After thesynthesis of new copies of viral proteins and genes, these componentsassemble into progeny virus particles, which then exit the cell(reviewed by Roizman and Palese, 1996). Different viral proteins play arole in each of these steps.

The influenza A virus is an enveloped negative-strand virus with eightRNA segments encapsidated with nucleoprotein (NP) (reviewed by Lamb andKrug, 1996). The eight single-stranded negative-sense viral RNAs (vRNAs)encode a total of ten to eleven proteins. The influenza virus life cyclebegins with binding of the hemagglutinin (HA) to sialic acid-containingreceptors on the surface of the host cell, followed by receptor-mediatedendocytosis. The low pH in late endosomes triggers a conformationalshift in the HA, thereby exposing the N-terminus of the HA2 subunit (theso-called fusion peptide). The fusion peptide initiates the fusion ofthe viral and endosomal membrane, and the matrix protein (M1) and RNPcomplexes are released into the cytoplasm. RNPs consist of thenucleoprotein (NP), which encapsidates vRNA, and the viral polymerasecomplex, which is formed by the PA, PB1, and PB2 proteins. RNPs aretransported into the nucleus, where transcription and replication takeplace. The RNA polymerase complex catalyzes three different reactions:synthesis of an mRNA with a 5′ cap and 3′ polyA structure, of afull-length complementary RNA (cRNA), and of genomic vRNA using the cDNAas a template. Newly synthesized vRNAs, NP, and polymerase proteins arethen assembled into RNPs, exported from the nucleus, and transported tothe plasma membrane, where budding of progeny virus particles occurs.The neuraminidase (NA) protein plays a crucial role late in infection byremoving sialic acid from sialyloligosaccharides, thus releasing newlyassembled virions from the cell surface and preventing the selfaggregation of virus particles. Although virus assembly involvesprotein-protein and protein-vRNA interactions, the nature of theseinteractions is largely unknown.

Although influenza B and C viruses are structurally and functionallysimilar to influenza A virus, there are some differences. For example,the M segment of influenza B virus encodes two proteins, M1 and BM2,through a termination-reinitiation scheme of tandem cistrons, and the NAsegment encodes the NA and NB proteins from a bicistronic mRNA.Influenza C virus, which has 7 vRNA segments, relies on splicedtranscripts to produce M1 protein; the product of the unspliced mRNA isproteolytically cleaved to yield the CM2 protein. In addition, influenzaC virus encodes a HA-esterase (HEF) rather than individual HA and NAproteins.

Spanning the viral membrane for influenza A virus are three proteins:hemagglutinin (HA), neuraminidase (NA), and M2. The extracellulardomains (ectodomains) of HA and NA are quite variable, while theectodomain domain of M2 is essentially invariant among influenza Aviruses. The M2 protein which possesses ion channel activity (Pinto etal., 1992), is thought to function at an early state in the viral lifecycle between host cell penetration and uncoating of viral RNA (Martinand Helenius, 1991; reviewed by Helenius, 1992; Sugrue et al., 1990).Once virions have undergone endocytosis, the virion-associated M2 ionchannel, a homotetrameric helix bundle, is believed to permit protons toflow from the endosome into the virion interior to disrupt acid-labileM1 protein-ribonucleoprotein complex (RNP) interactions, therebypromoting RNP release into the cytoplasm (reviewed by Helenius, 1992).In addition, among some influenza strains whose HAs are cleavedintracellularly (e.g., A/fowl plagues/Rostock/34), the M2 ion channel isthought to raise the pH of the trans-Golgi network, preventingconformational changes in the HA due to conditions of low pH in thiscompartment (Hay et al., 1985; Ohuchi et al., 1994; Takeuchi and Lamb,1994).

Cell Lines that can be Used in the Present Invention

Any cell, e.g., any avian or mammalian cell, such as a human, e.g., 293Tor PER.C6® cells, or canine, bovine, equine, feline, swine, ovine,rodent, for instance mink, e.g., MvLu1 cells, or hamster, e.g., CHOcells, non-human primate, e.g., Vero cells, or non-primate highervertebrate cells, e.g., MDCK cells, including mutant cells such as AX4cells, which support efficient replication of influenza virus can beemployed to isolate and/or propagate influenza viruses. Isolated virusescan be used to prepare a reassortant virus. In one embodiment, hostcells for vaccine production are continuous mammalian or avian celllines or cell strains. A complete characterization of the cells to beused, may be conducted so that appropriate tests for purity of the finalproduct can be included. Data that can be used for the characterizationof a cell includes (a) information on its origin, derivation, andpassage history; (b) information on its growth and morphologicalcharacteristics; (c) results of tests of adventitious agents; (d)distinguishing features, such as biochemical, immunological, andcytogenetic patterns which allow the cells to be clearly recognizedamong other cell lines; and (e) results of tests for tumorigenicity. Inone embodiment, the passage level, or population doubling, of the hostcell used is as low as possible.

In one embodiment, the cells are WHO certified, or certifiable,continuous cell lines. The requirements for certifying such cell linesinclude characterization with respect to at least one of genealogy,growth characteristics, immunological markers, virus susceptibilitytumorigenicity and storage conditions, as well as by testing in animals,eggs, and cell culture. Such characterization is used to confirm thatthe cells are free from detectable adventitious agents. In somecountries, karyology may also be required. In addition, tumorigenicitymay be tested in cells that are at the same passage level as those usedfor vaccine production. The virus may be purified by a process that hasbeen shown to give consistent results, before vaccine production (see,e.g., World Health Organization, 1982).

Virus produced by the host cell may be highly purified prior to vaccineor gene therapy formulation. Generally, the purification proceduresresult in extensive removal of cellular DNA and other cellularcomponents, and adventitious agents. Procedures that extensively degradeor denature DNA may also be used.

Influenza Vaccines

A vaccine of the invention includes an isolated recombinant influenzavirus of the invention, and optionally one or more other isolatedviruses including other isolated influenza viruses, one or moreimmunogenic proteins or glycoproteins of one or more isolated influenzaviruses or one or more other pathogens, e.g., an immunogenic proteinfrom one or more bacteria, non-influenza viruses, yeast or fungi, orisolated nucleic acid encoding one or more viral proteins (e.g., DNAvaccines) including one or more immunogenic proteins of the isolatedinfluenza virus of the invention. In one embodiment, the influenzaviruses of the invention may be vaccine vectors for influenza virus orother pathogens.

A complete virion vaccine may be concentrated by ultrafiltration andthen purified by zonal centrifugation or by chromatography. Virusesother than the virus of the invention, such as those included in amultivalent vaccine, may be inactivated before or after purificationusing formalin or beta-propiolactone, for instance.

A subunit vaccine comprises purified glycoproteins. Such a vaccine maybe prepared as follows: using viral suspensions fragmented by treatmentwith detergent, the surface antigens are purified, byultracentrifugation for example. The subunit vaccines thus containmainly HA protein, and also NA. The detergent used may be cationicdetergent for example, such as hexadecyl trimethyl ammonium bromide(Bachmeyer, 1975), an anionic detergent such as ammonium deoxycholate(Laver & Webster, 1976); or a nonionic detergent such as thatcommercialized under the name TRITON X100. The hemagglutinin may also beisolated after treatment of the virions with a protease such asbromelin, and then purified. The subunit vaccine may be combined with anattenuated virus of the invention in a multivalent vaccine.

A split vaccine comprises virions which have been subjected to treatmentwith agents that dissolve lipids. A split vaccine can be prepared asfollows: an aqueous suspension of the purified virus obtained as above,inactivated or not, is treated, under stirring, by lipid solvents suchas ethyl ether or chloroform, associated with detergents. Thedissolution of the viral envelope lipids results in fragmentation of theviral particles. The aqueous phase is recuperated containing the splitvaccine, constituted mainly of hemagglutinin and neuraminidase withtheir original lipid environment removed, and the core or itsdegradation products. Then the residual infectious particles areinactivated if this has not already been done. The split vaccine may becombined with an attenuated virus of the invention in a multivalentvaccine.

Inactivated Vaccines.

Inactivated influenza virus vaccines are provided by inactivatingreplicated virus using known methods, such as, but not limited to,formalin or β-propiolactone treatment. Inactivated vaccine types thatcan be used in the invention can include whole-virus (WV) vaccines orsubvirion (SV) (split) vaccines. The WV vaccine contains intact,inactivated virus, while the SV vaccine contains purified virusdisrupted with detergents that solubilize the lipid-containing viralenvelope, followed by chemical inactivation of residual virus.

In addition, vaccines that can be used include those containing theisolated HA and NA surface proteins, which are referred to as surfaceantigen or subunit vaccines.

Live Attenuated Virus Vaccines.

Live, attenuated influenza virus vaccines, such as those including arecombinant virus of the invention can be used for preventing ortreating influenza virus infection. Attenuation may be achieved in asingle step by transfer of attenuated genes from an attenuated donorvirus to a replicated isolate or reassorted virus according to knownmethods. Since resistance to influenza A virus is mediated primarily bythe development of an immune response to the HA and/or NA glycoproteins,the genes coding for these surface antigens come from the reassortedviruses or clinical isolates. The attenuated genes are derived from anattenuated parent. In this approach, genes that confer attenuationgenerally do not code for the HA and NA glycoproteins.

Viruses (donor influenza viruses) are available that are capable ofreproducibly attenuating influenza viruses, e.g., a cold adapted (ca)donor virus can be used for attenuated vaccine production. Live,attenuated reassortant virus vaccines can be generated by mating the cadonor virus with a virulent replicated virus. Reassortant progeny arethen selected at 25° C. (restrictive for replication of virulent virus),in the presence of an appropriate antiserum, which inhibits replicationof the viruses bearing the surface antigens of the attenuated ca donorvirus. Useful reassortants are: (a) infectious, (b) attenuated forseronegative non-adult mammals and immunologically primed adult mammals,(c) immunogenic and (d) genetically stable. The immunogenicity of the careassortants parallels their level of replication. Thus, the acquisitionof the six transferable genes of the ca donor virus by new wild-typeviruses has reproducibly attenuated these viruses for use in vaccinatingsusceptible mammals both adults and non-adult.

Other attenuating mutations can be introduced into influenza virus genesby site-directed mutagenesis to rescue infectious viruses bearing thesemutant genes. Attenuating mutations can be introduced into non-codingregions of the genome, as well as into coding regions. Such attenuatingmutations can also be introduced into genes other than the HA or NA,e.g., the M2 gene. Thus, new donor viruses can also be generated bearingattenuating mutations introduced by site-directed mutagenesis, and suchnew donor viruses can be used in the production of live attenuatedreassortants vaccine candidates in a manner analogous to that describedabove for the ca donor virus. Similarly, other known and suitableattenuated donor strains can be reassorted with influenza virus toobtain attenuated vaccines suitable for use in the vaccination ofmammals.

In one embodiment, such attenuated viruses maintain the genes from thevirus that encode antigenic determinants substantially similar to thoseof the original clinical isolates. This is because the purpose of theattenuated vaccine is to provide substantially the same antigenicity asthe original clinical isolate of the virus, while at the same timelacking pathogenicity to the degree that the vaccine causes minimalchance of inducing a serious disease condition in the vaccinated mammal.

The viruses in a multivalent vaccine can thus be attenuated orinactivated, formulated and administered, according to known methods, asa vaccine to induce an immune response in an animal, e.g., a mammal.Methods are well-known in the art for determining whether suchattenuated or inactivated vaccines have maintained similar antigenicityto that of the clinical isolate or high growth strain derived therefrom.Such known methods include the use of antisera or antibodies toeliminate viruses expressing antigenic determinants of the donor virus;chemical selection (e.g., amantadine or rimantidine); HA and NA activityand inhibition; and nucleic acid screening (such as probe hybridizationor PCR) to confirm that donor genes encoding the antigenic determinants(e.g., HA or NA genes) are not present in the attenuated viruses.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention, suitable forinoculation, e.g., nasal, parenteral or oral administration, compriseone or more influenza virus isolates, e.g., one or more attenuated orinactivated influenza viruses, a subunit thereof, isolated protein(s)thereof, and/or isolated nucleic acid encoding one or more proteinsthereof, optionally further comprising sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. The compositions can furthercomprise auxiliary agents or excipients, as known in the art. Thecomposition of the invention is generally presented in the form ofindividual doses (unit doses).

Conventional vaccines generally contain about 0.1 to 200 μg, e.g., 30 to100 μg, of HA from each of the strains entering into their composition.The vaccine forming the main constituent of the vaccine composition ofthe invention may comprise a single influenza virus, or a combination ofinfluenza viruses, for example, at least two or three influenza viruses,including one or more reassortant(s).

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and/or emulsions, which may containauxiliary agents or excipients known in the art. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Carriers or occlusive dressings can be used to increase skinpermeability and enhance antigen absorption. Liquid dosage forms fororal administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspendingliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, wetting agents, emulsifying and suspending agents, orsweetening, flavoring, or perfuming agents.

When a composition of the present invention is used for administrationto an individual, it can further comprise salts, buffers, adjuvants, orother substances which are desirable for improving the efficacy of thecomposition. For vaccines, adjuvants, substances which can augment aspecific immune response, can be used. Normally, the adjuvant and thecomposition are mixed prior to presentation to the immune system, orpresented separately, but into the same site of the organism beingimmunized.

Heterogeneity in a vaccine may be provided by mixing replicatedinfluenza viruses for at least two influenza virus strains, such as 2-20strains or any range or value therein. Vaccines can be provided forvariations in a single strain of an influenza virus, using techniquesknown in the art.

A pharmaceutical composition according to the present invention mayfurther or additionally comprise at least one chemotherapeutic compound,for example, for gene therapy, immunosuppressants, anti-inflammatoryagents or immune enhancers, and for vaccines, chemotherapeuticsincluding, but not limited to, gamma globulin, amantadine, guanidine,hydroxybenzimidazole, interferon-α, interferon-β, interferon-γ, tumornecrosis factor-alpha, thiosemicarbarzones, methisazone, rifampin,ribavirin, a pyrimidine analog, a purine analog, foscarnet,phosphonoacetic acid, acyclovir, dideoxynucleosides, a proteaseinhibitor, or ganciclovir.

The composition can also contain variable but small quantities ofendotoxin-free formaldehyde, and preservatives, which have been foundsafe and not contributing to undesirable effects in the organism towhich the composition is administered.

Pharmaceutical Purposes

The administration of the composition (or the antisera that it elicits)may be for either a “prophylactic” or “therapeutic” purpose. Whenprovided prophylactically, the compositions of the invention which arevaccines are provided before any symptom or clinical sign of a pathogeninfection becomes manifest. The prophylactic administration of thecomposition serves to prevent or attenuate any subsequent infection.When provided prophylactically, the gene therapy compositions of theinvention, are provided before any symptom or clinical sign of a diseasebecomes manifest. The prophylactic administration of the compositionserves to prevent or attenuate one or more symptoms or clinical signsassociated with the disease.

When provided therapeutically, a viral vaccine is provided upon thedetection of a symptom or clinical sign of actual infection. Thetherapeutic administration of the compound(s) serves to attenuate anyactual infection. When provided therapeutically, a gene therapycomposition is provided upon the detection of a symptom or clinical signof the disease. The therapeutic administration of the compound(s) servesto attenuate a symptom or clinical sign of that disease.

Thus, a vaccine composition of the present invention may be providedeither before the onset of infection (so as to prevent or attenuate ananticipated infection) or after the initiation of an actual infection.Similarly, for gene therapy, the composition may be provided before anysymptom or clinical sign of a disorder or disease is manifested or afterone or more symptoms are detected.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient mammal. Such an agent issaid to be administered in a “therapeutically effective amount” if theamount administered is physiologically significant. A composition of thepresent invention is physiologically significant if its presence resultsin a detectable change in the physiology of a recipient patient, e.g.,enhances at least one primary or secondary humoral or cellular immuneresponse against at least one strain of an infectious influenza virus.

The “protection” provided need not be absolute, i.e., the influenzainfection need not be totally prevented or eradicated, if there is astatistically significant improvement compared with a control populationor set of mammals. Protection may be limited to mitigating the severityor rapidity of onset of symptoms or clinical signs of the influenzavirus infection.

Pharmaceutical Administration

A composition of the present invention may confer resistance to one ormore pathogens, e.g., one or more influenza virus strains, by eitherpassive immunization or active immunization. In active immunization, anattenuated live vaccine composition is administered prophylactically toa host (e.g., a mammal), and the host's immune response to theadministration protects against infection and/or disease. For passiveimmunization, the elicited antisera can be recovered and administered toa recipient suspected of having an infection caused by at least oneinfluenza virus strain. A gene therapy composition of the presentinvention may yield prophylactic or therapeutic levels of the desiredgene product by active immunization.

In one embodiment, the vaccine is provided to a mammalian female (at orprior to pregnancy or parturition), under conditions of time and amountsufficient to cause the production of an immune response which serves toprotect both the female and the fetus or newborn (via passiveincorporation of the antibodies across the placenta or in the mother'smilk).

The present invention thus includes methods for preventing orattenuating a disorder or disease, e.g., an infection by at least onestrain of pathogen. As used herein, a vaccine is said to prevent orattenuate a disease if its administration results either in the total orpartial attenuation (i.e., suppression) of a clinical sign or conditionof the disease, or in the total or partial immunity of the individual tothe disease. As used herein, a gene therapy composition is said toprevent or attenuate a disease if its administration results either inthe total or partial attenuation (i.e., suppression) of a clinical signor condition of the disease, or in the total or partial immunity of theindividual to the disease.

A composition having at least one influenza virus of the presentinvention, including one which is attenuated and one or more otherisolated viruses, one or more isolated viral proteins thereof, one ormore isolated nucleic acid molecules encoding one or more viral proteinsthereof, or a combination thereof, may be administered by any means thatachieve the intended purposes.

For example, administration of such a composition may be by variousparenteral routes such as subcutaneous, intravenous, intradermal,intramuscular, intraperitoneal, intranasal, oral or transdermal routes.Parenteral administration can be accomplished by bolus injection or bygradual perfusion over time.

A typical regimen for preventing, suppressing, or treating an influenzavirus related pathology, comprises administration of an effective amountof a vaccine composition as described herein, administered as a singletreatment, or repeated as enhancing or booster dosages, over a period upto and including between one week and about 24 months, or any range orvalue therein.

According to the present invention, an “effective amount” of acomposition is one that is sufficient to achieve a desired effect. It isunderstood that the effective dosage may be dependent upon the species,age, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectwanted. The ranges of effective doses provided below are not intended tolimit the invention and represent dose ranges.

The dosage of a live, attenuated or killed virus vaccine for an animalsuch as a mammalian adult organism may be from about 10²-10¹⁵, e.g.,10³-10¹², plaque forming units (PFU)/kg, or any range or value therein.The dose of inactivated vaccine may range from about 0.1 to 1000, e.g.,10 to 100 μg, such as about 15 μg, of HA protein. However, the dosageshould be a safe and effective amount as determined by conventionalmethods, using existing vaccines as a starting point.

The dosage of immunoreactive HA in each dose of replicated virus vaccinemay be standardized to contain a suitable amount, e.g., 30 to 100 μg orany range or value therein, such as about 15 μg, or the amountrecommended by government agencies or recognized professionalorganizations. The quantity of NA can also be standardized, however,this glycoprotein may be labile during purification and storage.

The dosage of immunoreactive HA in each dose of replicated virus vaccinecan be standardized to contain a suitable amount, e.g., 1-50 μg or anyrange or value therein, or the amount recommended by the U.S. PublicHealth Service (PHS), which is usually 15 μg, per component for olderchildren □3 years of age, and 7.5 μg per component for older children <3years of age. The quantity of NA can also be standardized, however, thisglycoprotein can be labile during the processor purification and storage(Kendal et al., 1980; Kerr et al., 1975). Each 0.5-ml dose of vaccinemay contains approximately 1-50 billion virus particles, and preferably10 billion particles.

The invention will be described by the following nonlimiting examples.

Example I PB2 Incorporation Sequences

Most defective RNA segments of influenza A viruses retain 150 to 300nucleotides corresponding to the 5′ and 3′ ends of the respective genesegment (Duhaut et al., 1998; Jennings et al., 1983; Noble et al., 1995;and Odagiri et al., 1990), indicating that these 300 to 600 nucleotidesmay possess the structural features required for efficient genomepackaging. To identify the regions in the PB2, PB1, and PA vRNAs thatare critical for vRNA virion incorporation and virion formation,plasmids were generated in which the GFP gene is flanked by thenoncoding regions and portions of the coding regions derived from bothtermini [PB2(300)GFP(300), PB1(300)GFP(300), and PA(120)GFP(120)].Transfection of such a plasmid into 293T cells, together with expressionplasmids for the PB2, PB1, PA, and NP proteins (minimal components fortranscription and replication of vRNAs), resulted in the expression ofGFP in cells, indicating that the chimeric vRNAs were synthesized by thecellular RNA polymerase I and transcribed into mRNA by the viralproteins produced by the expression plasmids.

To calculate the vRNA virion incorporation efficiencies, the number ofvirions containing a test vRNA must be compared with the total number ofVLPs. The total number of VLPs could be determined by inoculating cellswith VLPs and then counting the number of cells expressing a giveninfluenza virus protection. To ensure that the number of infectious VLPsdetermined by this method was not drastically affected by the viral geneproduct selected as a marker, we determined the number of cellsexpressing either HA or NP. Because we were testing the incorporationefficiencies of the PB2, PB1, and PA vRNAs, helper virus was needed toprovide functional polymerase proteins. To distinguish between the HAand NP proteins expressed from out test VLPs (derived from WSN virus)and those expressed from the helper PR8 virus, we used antibiotics thatrecognize the WSN HA and NP proteins, but not their PR8 viruscounterparts.

To establish a system that allowed the assessment of the number of VLPsgenerated, 293T cells were transfected with a plasmid for thetranscription of a test vRNA (derived from the PB2, PB1, or PA segment),7 plasmids for the production of the remaining vRNAs, and 10 expressionplasmids for the expression of the viral proteins (i.e., PB2, PB1, PA,HA, NP, NA, M1, M2, NS1, and NS2). Forty-eight hours later,VLP-containing supernatants derived from transfected cells were mixedwith PR8 helper virus and used to infect MDCK cells. Twelve hourspostinfection, the number of cells that expressed either HA or NPprotein were determined. For all three vRNAs, the numbers of HA- orNP-expressing cells differed by less than a factor of 3; for example,using the PB2(300)GFP(300) test vRNA, 240,800 HA-expressing cells versus353,200 NP-expressing cells were detected. Therefore, for the subsequentexperiments, the number of HA-expressing cells was employed as anindicator of the efficiency of infectious virion formation. Theincorporation efficiencies of test vRNAs were thus calculated bydividing the number of GFP-expressing cells (as a marker for the testvRNA) by the sum of the number of HA-expressing cells (as a marker forthe number of virions) plus the number of GFP-expressing cells.

Sequences in the coding region of the PB2 vRNA affect infectious virionformation and vRNA virion incorporation. To delineate the sequences inthe PB2 vRNA that are critical for virion formation and/or vRNA virionincorporation, a series of plasmids was generated for the production ofPB2 vRNAs that express GFP and contain portions of the PB2 coding regionderived from both termini (FIG. 1), in addition to the noncoding regionsof the PB2 vRNA (FIG. 1). The numbers of VLPs and the incorporationefficiencies of the test vRNAs were determined as described above.

With PB2(300)GFP(300), which contains 300 nucleotides corresponding tothe 5′ and 3′ coding regions of the PB2 vRNA, about 2.8×10⁶ VLPs per mLwere detected. Stepwise deletion of the coding sequences at the 3′ endof the vRNA (referred to as the 3′ coding region) had only moderateeffects on the efficiency of VLP production; PB2(0)GFP(120), which lacksthe entire coding region of the 3′ end, yielded about 1×10⁶ VLPs/mL.Deletion of the coding sequences at the 5′ end of the vRNA (referred toas the 5′ coding region) [PB2(120)GFP(0)], however, reduced VLPproduction by 98% of that of PB2(300)GFP(300) and yielded a number ofVLPs comparable to that obtained in the absence of the PB2 vRNA[PB2(−)]. This result suggests that sequences in the 5′ coding region ofthe PB2 vRNA are critical for the efficient generation of infectiousvirions. Further analysis revealed that 30 nucleotides of the 5′ codingregion are critical for this effect [compare the numbers of VLPs forPB2(120)GFP(0) and PB2(120)GFP(30)].

With regard to the efficiencies of vRNA virion incorporation.PB2(300)GFP(300), 54.7% of the VLPs contained the PB2(300)GFP(300) testvRNA, indicating that the 300 terminal nucleotides at both ends aresufficient for virion incorporation. To achieve the incorporationefficiencies observed for wild-type segments, internal PB2 codingsequences would likely be required. Stepwise deletion of nucleotides inthe 3′ coding region of the PB2 vRNA had only moderate effects provided30 or more nucleotides were retrained; the deletion of these remaining30 nucleotides, however, reduced the virion incorporation efficiency to29.5% for PB2(0)GFP(120), demonstrating that this region is importantfor the efficient incorporation of the PB2 vRNA into virions. For PB2vRNAs that lack a functional packaging sequence in the 3′ coding region,sequences in the 5′ coding region do contribute to virion incorporation,as exemplified by the inability of the PB2(0)GFP(0) test vRNA to beincorporated.

Deletions in the 5′ coding region only, by contrast, had no effect onincorporation efficiencies, as demonstrated by a 75.7% incorporationrate for PB2(120)GFP(0). Thus while the use of this test vRNA produced avery low number of infectious VLPs, the test vRNA was efficientlyincorporated into these particles. This finding suggests that sequencesin the PB2 vRNA are involved in two biologically distinct processes:efficient infectious virion formation (a function residing in the 5′coding region) and efficient vRNA incorporation into particles (afunction primarily residing in the 3′ coding region).

The difference in packaging efficiencies could reflect differences intranscription levels of the test vRNAs in 293T cells. To exclude thispossibility, the levels of PB2(0)GFP(0) and PB2(120)GFP(120) inplasmid-transfected 293T cells were examined using real-time PCR. Theamount of PB2(0)GFP(0) vRNA was 52% of that of PB2(120)GFP(120);however, this difference is unlikely to explain the 99% reduction in VLPgeneration and the abrogation of vRNA virion incorporation.

PB2 vRNA is more critical for efficient infectious virion generationthan the PB1 or PA vRNA. Thus, a hierarchy may exist in which the PB2vRNA is critical for the efficient virion incorporation of other vRNAs,while the omission of other segments is tolerable to some extent.

Omission of the PB2 vRNA resulted in an about 30-fold reduction in VLPproduction, whereas omission of the other vRNA segments resulted in 1.4-to 5.1-fold reductions. These results provided further proof of ahierarchy among the vRNA segments with respect to the importance of theindividual vRNAs for the incorporation of the other vRNA segments.

Example II

The stable expression of a foreign gene in a replication-incompetentinfluenza virus allows for the effective tracking of the manipulatedvirus. In pursuit of a biologically contained foreign gene-expressingvirus with extensive applications in the field of virology, the PB2protein, an influenza viral polymerase subunit that forms part of thetrimeric viral RNA-dependent RNA polymerase that is essential for virusreplication was selected. The partial coding sequences of the 3′ and 5′ends of the PB2 viral RNA (vRNA) confer its more critical role inefficient infectious virion generation relative to the other vRNAs inthe vRNA hierarchy (Example I and Muramoto et al., 2006). This findingsuggests that a PB2-knock out (PB2-KO) influenza virus harboring areporter gene flanked by the coding and non-coding sequences of the PB2vRNA would replicate only in PB2-expressing cells while stablyexpressing the reporter gene.

A cell line that stably expresses PB2 protein was established and usedto characterize a PB2-KO virus that possesses the GFP gene. Thepotential for various virus strain-derived HA and NA genes, as well asother reporter genes, to be accommodated by the PB2-KO virus, was alsoinvestigated. Further, the PB2-KO virus was employed as a platform toscreen neutralizing antibodies against 2009 pandemic viruses.

Methods

Cells.

293T human embryonic kidney cells (a derivative of the 293 line intowhich the gene for simian virus 40 T antigen was inserted (DuBridge etal., 1987)) were maintained in Dulbecco's modified Eagle medium (Lonza)supplemented with 10% fetal calf serum (Invitrogen). Madin-Darby caninekidney (MDCK) cells were maintained in minimum essential medium (MEM;Invitrogen) supplemented with 5% newborn calf serum (NCS; Sigma, St.Louis, Mo.). AX4 cells, derived from MDCK cells and transfected with thecDNA of human 2,6-sialyltransferase (Hatakeyama et al., 2005), weremaintained in 5% NCS/MEM+puromycin (2 μg/mL). AX4/PB2 cells (AX4 cellsstably expressing the PB2 protein derived from A/Puerto Rico/8/34 (H1N1,PR8), established by transduction with a retroviral vector, see theResults section) were maintained in 5% NCS/MEM+puromycin (2μg/mL)+blasticidin (10 μg/mL). All cells were maintained at 37° C. in 5%CO₂.

Reverse Genetics and Virus Propagation.

Reverse genetics was performed with plasmids that contained the cDNAs ofthe PR8 viral genes between the human RNA polymerase 1 promoter and themouse RNA polymerase 1 terminator (referred to as Pol1 plasmids) andeukaryotic protein expression plasmids (NP, PA, PB1, and PB2) under thecontrol of the chicken β-actin promoter (Niwa et al., 1991), asdescribed in Neumann et al. (1999). Briefly, the wild-type PR8 virus wasengineered by using the eight previously produced wild-type constructsderived from PR8 (Horimoto et al., 2007); whereas the PB2-KO mutant wascomprised of pPolIPB2(120)GFP(120) (FIG. 3A) (Muramoto et al., 2006) andthe remaining seven segmental PolI plasmids. The pPolIPB2(120)GFP(120)plasmid contains the A/WSN/33 (H1N1, WSN)-derived 3′ PB2 non-codingregion, 120 nucleotides that correspond to the PB2 coding sequence atthe 3′ end of the vRNA followed by the GFP coding sequence, 120nucleotides that correspond to the PB2 coding sequence at the 5′ end ofthe vRNA, and finally the 5′ PB2 non-coding region (Muramoto et al.,2006). Likewise, pPolIPB2(120)Fluc(120) and pPolIPB2(120)Rluc(120) wereconstructed to generate PB2-KO viruses possessing the firefly luciferase(Fluc) or Renilla luciferase (Rluc) genes, respectively. The eight PolIplasmids and protein expression plasmids were mixed with thetransfection reagent TransIT-293 (Minis), incubated at room temperaturefor 15 minutes, and added to 10⁶ 293T cells cultured in Opti-MEM 1(Invitrogen). Forty-eight hours post-transfection, the supernatantcontaining wild-type PR8 or PB2-KO virus was harvested and propagated in10-day-old embryonated chicken eggs or AX4/PB2 cells, respectively.Wild-type CA04 was also generated by using reverse genetics, asdescribed in Yamada et al. (2010), and propagated in MDCK cells. Thepropagated viruses were titrated by using plaque assays in MDCK cells todetermine plaque-forming units (PFU) of virus.

Immunofluorescence Staining of the PB2 Protein.

Confluent AX4 and AX4/PB2 cells seeded in 35 mm glass bottom dishes(Asahi Techno Glass) were fixed in phosphate buffered saline (PBS)containing 4% paraformaldehyde (Wako Pure Chemical Industries Ltd) andpermeabilized with 0.1% Triton X-100. Cells were incubated with ananti-PB2 antibody clone 18/1 (Hatta et al., 2000) and further incubatedwith an Alexa Fluor 594-labeled anti-mouse secondary antibody(Invitrogen) in Hoescht 33342 (Invitrogen). Samples were observed undera confocal laser microscope (LSM510META; Carl Zeiss).

Reverse Transcription-PCR (RT-PCR).

To detect PB2 mRNA in AX4/PB2 cells, total RNA was extracted by using anRNeasy RNA extraction kit (Qiagen Sciences). Viral RNAs were isolatedfrom virions by using a QIAmp viral RNA mini kit (Qiagen Sciences).Reverse transcription and cDNA synthesis were performed by usingoligo(dT) primer and SuperScript III reverse transcriptase (Invitrogen).RT-minus samples were prepared as negative controls. The synthesizedcDNA was amplified by use of PCR with Phusion PCR polymerase (Finnzymes)and PB2-specific primers. Primer sequences are as follows:

forward primer, (SEQ ID NO: 9) ATGGAAAGAATAAAAGAACTACGA,  andreverse primer (SEQ ID NO: 16) GCCACAATTATTGCTTCGGC.

Growth Kinetics and Virus Titration.

To determine virus growth rates, triplicate wells of confluent AX4 orAX4/PB2 cells were infected at a multiplicity of infection (MOI) of0.001. After 1 hour of virus adsorption, cells were washed in MEMcontaining 0.3% BSA, overlaid with MEM containingL-(tosylamido-2-phenyl)ethyl chloromethyl ketone (TPCK)-treated trypsin(0.5 μg/mL). Supernatants were collected every 12 hours for three daysand assayed for infectious virus in plaque assays in AX4/PB2 cells.

Immunostaining.

To assess the stability of the GFP reporter gene incorporation in thePB2-KO virus, AX4/PB2 cells were infected with various PB2-KO virusdilutions (undiluted to) 10⁻¹⁰. The supernatant from the second to lastwell in which a cytopathic effect (CPE) was observed, was harvested anddiluted for subsequent infections. Supernatants from five rounds ofvirus passaging were subjected to standard virus plaque assays. Once thenumber of plaques formed was counted, the agar was removed and wellscontaining plaques were fixed with 100% methanol for 30 minutes. Wellswere then washed with PBS and incubated with a monoclonal anti-GFPantibody (clone GFP-20; Sigma-Aldrich) at room temperature for 1 hour.Immunohistochemical staining was performed by using a biotinylatedanti-mouse antibody according to the Vectastain Elite ABC kitinstructions (Vector Laboratories). GFP-positive plaques were visualizedby using Sigma Fast 3,3′-Diaminobenzidine tablets (Sigma), and thenumber of GFP-positive plaques was calculated as a percentage of thetotal number of plaques that formed in the respective wells.

Immunofluorescent Staining for HA Protein.

GFP-encoding PB2-KO virus possessing HA and NA vRNAs derived from PR8,WSN, A/California/04/09 (CA04), or A/Vietnam/1203/04 (VN1203) weregenerated by using reverse genetics, as described above, and propagatedin AX4/PB2 cells. The multiple basic amino acid residues in the VN1203HA cleavage site were replaced with a non-virulent type cleavagesequence. Confluent AX4/PB2 cells were infected with these viruses at anMOI of 0.2-1. At 16 hours post-infection, cells were fixed with 4%paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100. Cellswere then incubated with an anti-WSN HA antibody (WS 3-54), an anti-CA04HA antibody (IT-096; eENZYME), and an anti-H5 HA antibody (VN04-10;Rockland Immunochemicals Inc.) and then further incubated with an AlexaFluor 594-labeled anti-mouse secondary antibody. Samples were observedunder a fluorescence microscope.

Luciferase Assay.

Cells infected with PB2-KO virus encoding Fluc or Rluc gene weresubjected to a luciferase assay by using a dual-luciferase reporterassay system (Promega) at 8 hours post-infection according to themanufacturer's instructions. Fluc and Rluc activities were measured witha microplate reader Infinite M1000 (Tecan).

Microneutralization Assay.

Sera were collected from two ferrets infected with 10⁶ PFU of wild-typeCA04 three weeks post-infection and from two uninfected ferrets.Two-fold serial dilutions of receptor-destroying enzyme (DENKA SEIKENCO., LTD)-treated ferret sera were mixed with 100 PFU of wild-type CA04or Rluc-encoding PB2-KO virus possessing CA04-derived HA and NA vRNAs(CA04/PB2-Rluc). After incubation at 37° C. for 1 hour, wild-type AX4 orAX4/PB2 cells were inoculated with the wild-type virus- or PB2-KOvirus-serum mixtures, respectively, in triplicate wells and incubatedfor three days or 24 hours for the wild-type virus or PB2-KO virus,respectively. The neutralization activity of the ferret sera wasdetermined on the basis of the CPE observed under the microscope or theRluc activity as measured by using the Renilla luciferase assay system(Promega) for the wild-type virus or PB2-KO virus, respectively.

Results

Characterization of the PR8/PB2-GFP Virus.

To establish a cell line that stably expresses PB2 protein, AX4 cells,which are human 2,6-sialyltransferase overexpressing Madin-Darby caninekidney (MDCK) cells that allow better replication of clinical influenzaisolates compared with wild-type MDCK cells (Hatakeyama et al., 2005)were transduced, with a retroviral vector that possessed the cDNA of theA/Puerto Rico/8/34 (H1N1, PR8) PB2 protein followed by an internalribosome entry site sequence derived from the encephalomyocarditis virusand the blasticidin resistance gene. A blasticidin-resistant cell clonewas designated as AX4/PB2. To confirm the expression of mRNA for the PB2protein in AX4/PB2 cells, total RNAs were extracted from AX4/PB2 andwild-type AX4 cells and subjected to RT-PCR with PB2-specific primers.PB2 mRNA was detected in AX4/PB2 cells but not in wild-type AX4 cells(FIG. 3B). To further validate the expression of the PB2 protein inAX4/PB2 cells, immunofluorescence staining of AX4/PB2 cells wasperformed by using a PB2-specific monoclonal antibody. Fluorescentsignals were detected in AX4/PB2 cells and in some of the PB2 proteinexpression plasmid-transfected AX4 cells (which served as a positivecontrol), whereas no signal was detected in wild-type AX4 cells (FIG.3C). These results indicate that AX4/PB2 cells stably express the PB2protein.

To investigate whether PB2-expressing cells support PB2-KO virusreplication, a PR8-based PB2-KO virus possessing PB2(120)GFP(120) vRNA(FIG. 3A), designated as PR8/PB2-GFP (Table 1), was generated by andused to infect AX4/PB2 and wild-type AX4 cells (FIG. 3D). Although noinfectious virus was detected in wild-type AX4 cells, replication ofPR8/PB2-GFP virus in AX4/PB2 cells was comparable to that of wild-typePR8 (FIG. 3D). These results indicate that the replication of PB2-KOvirus is restricted to PB2-expressing cells.

TABLE 1 Origin of: NA Remaining Virus HA gene gene PB2 gene genesWild-type PR8 PR8^(*) PR8 PR8 PR8/PB2-GFP PR8 PR8 PB2(120)GFP(120)^(∥)PR8ΔPB2 PR8 PR8 -^(¶) WSN/PB2-GFP WSN^(†) WSN PB2(120)GFP(120)CA04/PB2-GFP CA04^(‡) CA04 PB2(120)GFP(120) PR8 VN1203/PB2-GFPVN1203^(§) VN1203 PB2(120)GFP(120) PR8/PB2-Rluc PR8 PR8PB2(120)Rluc(120)^(∥) PR8/PB2-Fluc PR8 PR8 PB2(120)Fluc(120)^(∥)CA04/PB2-Rluc CA04 CA04 PB2(120)Rluc(120) ^(*)PR8, A/Puerto Rico/8/34(H1N1). ^(†)WSN, A/WSN/33 (H1N1). ^(‡)CA04, A/California/04/09 (H1N1).^(§)VN1203, A/Vietnam/1203/04 (H5N1). The multiple basic amino acidresidues in the HA cleavage site (RERRRKKR↓G) were replaced with anon-virulent type cleavage sequence (RETR↓G). ^(∥)PB2(120)GFP(120),PB2(120)Rluc(120), and PB2(120)Fluc(120). GFP, firefly luciferase, andRenilla luciferase genes, respectively, flanked by the 3′ and 5′non-coding sequences and 120 bases of the 3′ and 5′ coding sequences ofthe PB2 gene. ^(¶)-, not applicable.

The stability of the reporter gene in PB2-KO virus was ascertained byserial passaging of PR8/PB2-GFP virus in AX4/PB2 cells. GFP-expressingplaques versus total plaques formed were calculated to determine thepercentage of plaque-forming viruses expressing the GFP reporter gene(Table 2); after five serial passages, 80%-90% of the plaque-formingviruses expressed GFP. PB2-KO virus failed to form plaques in wild-typecells even after five serial passages in AX4/PB2 cells, indicating thatreversion of PB2-KO virus to a replication-competent genotype byrecombination between the PB2-GFP vRNA and the cell-expressed PB2 mRNAis unlikely. An attempt was made to rescue a PB2 gene-deficient viruspossessing seven vRNA segments (PR8ΔPB2, Table 1); however, neithercytopathic effect (CPE) nor NP protein expression were observed inAX4/PB2 or wild-type AX4 cells inoculated with the transfectantsupernatant for PR8ΔPB2 (data not shown). These results highlight theimportance of the PB2 vRNA for efficient generation of infectiousvirions (Muramoto et al., 2006).

TABLE 2 Genetic stability of PB2-KO virus. Ratio of GFP-positiveplaques^(*) from viruses in: Passage 1 Passage 2 Passage 3 Passage 4Passage 5 Exp. 1 100% 90% 90% 100% 80% Exp. 2 100% 100%  90% 100% 90%Exp. 3 100% 90% 90% 100% 90% ^(*)The respective viral supernatants weresubjected to standard virus plaque assays in confluent AX4/PB2 cells.Ten plaques were marked per well, which were then subjected to theimmunodetection assay by using an anti-GFP antibody to detectGFP-expressing viral plaques. The percentage of plaques that stainedbrown among all plaques is presented. The results of three independentexperiments (Exp.) are shown.

Functional Expression of Different HA and NA Genes in PB2-KO Virus.

Two surface glycoproteins on influenza A virions, hemagglutinin (HA) andneuraminidase (NA), are the main protective antigens (Wright et al.,2007). In particular, HA mediates cell attachment; therefore, anantibody against HA is crucial for virus neutralization. It was testedwhether the relevant glycoproteins of a PR8 virus-based PB2-KO viruscould be replaced with those of other virus strains. To this end,GFP-encoding PB2-KO viruses were generated by substituting PR8HA and NAvRNAs with those derived from a laboratory H1N1 strain A/WSN/33(WSN/PB2-GFP), a 2009 pandemic (H1N1) strain A/California/04/2009(CA04/PB2-GFP), or a highly pathogenic H5N1 strain A/Vietnam/1203/2004(VN1203/PB2-GFP) (Table 1). AX4/PB2 cells were infected with theseviruses and subjected to an immunofluorescence assay with variousanti-HA monoclonal antibodies. In the GFP-positive virus-infected cells,virus strain-specific HA expression was detected (FIG. 4). It wasconfirmed by sequencing that the corresponding NA vRNAs wereincorporated into virions (data not shown). These results indicate thatthe HA and NA genes of other influenza viruses can also be accommodatedin the PB2-KO virus and hence be expressed in virus-infected cells.

Expression of Alternative Reporter Genes in PB2-KO Virus.

The activity of the luciferase reporter gene is readily quantifiableand, therefore, its incorporation into PB2-KO virus should allow one tomeasure virus replication based on luciferase activity. To test thispossibility, PB2-KO viruses were generated that possessed either thefirefly (PR8/PB2-Fluc) or Renilla (PR8/PB2-Rluc) luciferase gene in thePB2 vRNA (Table 1). AX4/PB2 and wild-type AX4 cells were infected withthese viruses at various multiplicities of infection (MOIs) andsubjected to a luciferase assay at 8 hours post-infection. Invirus-infected AX4/PB2 cells, Fluc and Rluc activities were detected ina dose-dependent manner; viruses infected at an MOI of 0.1 and 0.001were adequate for detecting significant Fluc and Rluc activities,respectively (FIG. 5A). By contrast, to detect significant GFP intensityin virus-infected cells, we needed to infect PR8/PB2-GFP at an MOI of 1or higher (FIG. 5B). These results indicate that the Fluc and Rluc genescan be accommodated in PB2-KO virus and represent a more quantitativeindicator for virus replication than does the GFP gene. Wild-type AX4cells infected with PR8/PB2-Fluc and PR8/PB2-Rluc also exhibiteddetectable Fluc and Rluc activities, respectively, at an MOI of morethan 1 for PR8/PB2-Fluc or 0.01 for PR8/PB2-Rluc, although the activityof both reporter genes was more than 10-fold lower than that detected inAX4/PB2 cells (FIG. 5A). Since the PB2 protein was not provided in transto the wild-type AX4 cells, the expression of these reporter genessuggests that viral ribonucleoproteins (i.e., PB2, PB1, PA, and NP)derived from incoming viruses mediate the transcription of the PB2 vRNAof PB2-KO virus at a significantly high level in wild-type AX4 cells.

PB2-KO Virus-Based Microneutralization.

Biologically contained, reporter gene-expressing influenza viruses havethe potential to supersede conventional virus replication evaluationsystems in part because of the ability to quantitate growth viaplatereader assays. The neutralization activity of antisera is typicallydetermined by using conventional microneutralization assays (Itoh etal., 2009; Kobasa et al., 2004), which allow the detection ofneutralizing antibodies based on the presence or absence of virusinfection-induced CPE or of virus antigens, as detected by using anenzyme-linked immunosorbent assay. To use PB2-KO virus to detectneutralizing antibodies against 2009 pandemic viruses, a PB2-KO viruswas generated that possessed the Rluc gene-encoding PB2 vRNA andA/California/04/2009-derived HA and NA vRNAs (CA04/PB2-Rluc).CA04/PB2-Rluc (100 PFU) was mixed with serially diluted antiseracollected from CA04-infected ferrets and incubated at 37° C. for 1 hour.Sera from uninfected ferrets served as negative control. The virus-seramixtures were used to inoculate AX4/PB2 cells. At 24 hourspost-infection, Rluc activity in cells was measured by using the Renillaluciferase assay system (Promega). To compare the detection sensitivity,the same antisera were also tested for neutralization activity by usinga CPE-based conventional microneutralization assay with wild-type CA04and wild-type AX4 cells. In the PB2-KO virus-based assay, 1:1280- and1:640-diluted ferret sera induced a significant decrease in Rlucactivity in virus-infected cells (FIG. 6). By contrast, the neutralizingtiters of the same ferret sera as determined in the conventionalmicroneutralization assay were 160 and 80 (data not shown). Theseresults indicate that the PB2-KO virus coupled with PB2-expressing cellsoffer a neutralizing antibody detection method that is more sensitivethan the conventional microneutralization assay.

Discussion

Here, it is demonstrated that PB2-KO influenza viruses arereplication-incompetent in wild-type cells, but undergo multiplereplication cycles in PB2 protein-expressing cells (FIG. 3D). Inaddition, reporter genes flanked by the PB2 vRNA packaging signals werestably maintained in progeny viruses (Table 1) and expressed invirus-infected cells (FIGS. 4 and 5). It was also confirmed thatdifferent virus strain-derived HA and NA genes were accommodated byPB2-KO viruses (FIG. 4). These results indicate that PB2-KO viruses havebroad potential use throughout the field of influenza virology.

As a practical application, a PB2-KO virus-based microneutralizationassay was developed and used to detect neutralizing antibodies againstthe 2009 pandemic virus (FIG. 6). This PB2-KO virus-based assay provedto be more sensitive than the conventional microneutralization assay interms of neutralizing antibody detection. The use ofreplication-incompetent PB2-KO viruses as a screening platform (FIGS. 3Cand 3D) may enable the detection of neutralizing antibodies againsthighly pathogenic viruses such as H5N1 and 1918 strains, which normallymust be handled in BSL3 facilities and under biosafety level 2containment, although an additional layer of biosafety (e.g.,modification of the amino acid sequence of the HA cleavage site) wouldbe required. Kong et al. (2006) previously developed a neutralizingantibody screening system based on influenza HA-pseudotypedlentiviruses, which also allows the detection of neutralizing antibodiesagainst the bio safety level 3 agents. However, these lentiviruses donot express influenza viral neuraminidase, which, along with HA, has thepotential to induce neutralizing antibodies (Nayak et al., 2010);therefore, the PB2-KO virus-based assay should more accurately reflectthe neutralizing antibody titers. Although cells that stably expressreporter gene-encoding influenza vRNA have also been shown to allow thesensitive detection of neutralizing antibodies (Hossain et al., 2010; Liet al., 2009), infectious viruses are required for these recombinantcell-based assays.

Another potential application of the PB2-KO virus is its use as a novelinfluenza vaccine, which we believe is feasible for the followingreasons. First, PB2-KO virus generates high titers (>10⁸ PFU/mL) in theAX4/PB2 cell line (FIG. 3D); second, the fact that HA and NA proteinscan be expressed (FIG. 4) demonstrates that PB2-KO virus is customizableto encode desired antigens; third, the vRNA transcription that occurs inPB2-KO virus-infected cells (FIG. 5A) may stimulate cellular innateimmunity by producing double-stranded RNA; and fourth, the stablemaintenance of a foreign gene inserted in the PB2 vRNA (Table 2) couldserve as a carrier of an additional antigen, enabling the engineering ofPB2-KO as a safe multi-valent vaccine.

To date, several recombinant influenza viruses that lack a particularviral protein have been shown to replicate comparably to wild-type virusin cell culture when the missing protein is provided in trans.M2-lacking influenza virus efficiently replicates in M2-expressing cellsand has demonstrated potential as a live attenuated vaccine (Watanabe etal., 2009). A distinct advantage of the PB2-KO virus over its M2counterpart is that the former is replication-incompetent in normalcells and, thus, safer. Further, it remains unknown whether a foreigngene encoded in the M2 protein-coding region can be incorporated intoprogeny viruses and expressed in virus-infected cells.

Martínez-Sobrido et al. (2010) developed an improved screening assay forthe rapid detection of neutralizing antibodies by using influenza viruspossessing the GFP gene flanked by the HA vRNA packaging signals.Although this HA-KO virus underwent multiple replication cycles only incells that expressed the HA protein, the stability of reporter genes inthis HA-KO virus was not tested in the study. In fact, an HAvRNA-deficient virus possessing seven vRNA segments underwent multiplerounds of replication in HA-expressing MDCK cells (data not shown) incontrast to the PB2 vRNA-deficient PR8ΔPB2 virus (see above), suggestingthat the reporter gene-encoding HA vRNA in HA-KO virus could be easilydropped during replication in HA-expressing cells. Areplication-competent virus that possesses the GFP gene in its NA vRNAhas also been used to detect neutralizing antibodies (Rimmelzwaan etal., 2011). An in trans bacterial sialidase improved the restrictedreplication of this NA-KO virus and allowed reasonable virus titerrecovery; however, the reporter gene stability of the NA-KO virusremains uncertain.

More recently, GFP gene-possessing replication-competent influenzaviruses have been generated by using recombinant NS (Manicassamy et al.,2010) or NA (Li et al., 2010) genes. Although these viruses havepotential as research tools, their replicability raises biosafetyissues, which are not a concern with the PB2-KO virus. Overall, the factthat the PB2-KO virus produced in this study stably expresses a foreigngene and is replication-incompetent makes it ideal in terms ofreliability and bio safety.

In conclusion, a biologically contained foreign gene-expressinginfluenza virus was generated by replacing the viral PB2 gene withreporter genes. The replication of the virus was restricted to cellsthat expressed the PB2 protein in trans. The reporter gene was stablyinherited in progeny viruses during replication in PB2-expressing cells.Various HA, NA, and reporter genes were accommodated in the PB2-KOvirus. This virus, therefore, shows promise in terms of its numerousapplications for basic and applied studies of influenza virus.

Example III

The PB2 protein of influenza viruses is an essential component of thetrimeric viral RNA-dependent RNA polymerase subunit. PB2 is implicatedin the regulation of host antiviral immune pathways and hence virulenceand plays a major role in the incorporation of other individual vRNAsegments (Muramoto et al., 2006). Example II describes a PB2-knock-out(PB2-KO) influenza virus that harbors reporter genes, such as the greenfluorescent protein (GFP) in the coding region of its PB2 viral RNA(vRNA). The replication of the PB2-KO virus was restricted to only acell line stably expressing the PB2 protein, yielding a high titre of>10⁸ PFU/mL. Moreover, during replication PB2 vRNA encoding the reportergene was stably incorporated into progeny virions and retained throughsequential passages. Also, HA and NA vRNA of any influenza virus couldbe accommodated in the PB2-KO virus and be expressed in virus-infectedcells. Therefore, the PB2-KO virus can be tailored to encode desiredcombinations of HA and NA that are the main influenza antigens,suggesting that the PB2-KO virus can be used as a multivalent vaccineplatform. Here, we tested the vaccine potential of the PB2-KO virus byimmunizing mice, examining antibody response and protective efficacy inmice.

Materials and Methods

Cells.

293 and 293T (a derivative of the 293 line into which the gene forsimian virus 40 T antigen was inserted (DuBridge et al., 1987) humanembryonic kidney cells were maintained in Dulbecco's modified Eaglemedium (Lonza, Basel, Switzerland) supplemented with 10% fetal calfserum (Invitrogen, Carlsbad, Calif.). Madin-Darby canine kidney (MDCK)cells were maintained in minimum essential medium (MEM) (Invitrogen)supplemented with 5% newborn calf serum (NCS) (Sigma, St. Louis, Mo.).AX4 cells, which are an MDCK-derived cell line with enhanced expressionof human-type receptors for influenza virus and were produced by stabletransfection of a plasmid expressing the human α-2,6-sialyltransferase(Hatakeyama et al., 2005), were maintained in 5% NCS-MEM supplementedwith puromycin (2 μg/mL). AX4/PB2 cells (AX4 cells stably expressing thePB2 protein derived from A/Puerto Rico/8/34 [H1N1, PR8], established bytransduction with a retroviral vector (Ozawa et al., 2011) weremaintained in 5% NCS-MEM supplemented with puromycin (2 μg/mL) andblasticidin (10 μg/mL). All cells were maintained in a humidifiedincubator at 37° C. in 5% CO₂.

Plasmid-driven reverse genetics. The wild-type PR8 and PB2-KO virusesused in this study were engineered by using reverse genetics, aspreviously described (Neumann et al., 1999). For expression of viral RNA(vRNA), plasmids contained the cloned cDNAs of PR8 genes between thehuman RNA polymerase I promoter and the mouse RNA polymerase Iterminator (referred to as PolI plasmids). A plasmid[pPolIPB2(120)GFP(120)] was constructed to replace the PolI plasmidencoding the PB2 segment and contained the A/WSN/33(H1N1)-derived 3′ PB2noncoding region, 120 nucleotides that correspond to the PB2-codingsequence at the 3′ end of the vRNA followed by the GFP-coding sequence,120 nucleotides that correspond to the PB2-coding sequence at the 5′ endof the vRNA, and finally the 5′ PB2 noncoding region (Muramoto et al.,2006). To generate the PB2-KO virus, pPolIPB2(120)GFP(120) and theremaining 7 PolI plasmids were cotransfected into 293T cells along witheukaryotic protein expression plasmids for PB2, PB1, PA, and NP derivedfrom A/WSN/33 by use of the TransIT 293 transfection reagent (Mirus,Madison, Wis.), following the manufacturer's instructions. At 48 hourspost-transfection, the supernatants containing the PR8 or PB2-KO viruswere harvested and inoculated into 10-day-old embryonated chicken eggsor AX4/PB2 cells, respectively. Both viruses were titrated by use ofplaque assay with AX4/PB2 cells.

Preparation of Formalin-Inactivated Virus.

Egg-propagated PR8 viruses were concentrated and purified byultracentrifugation of the infected allantoic fluid through a 10% to 50%sucrose density gradient and resuspended in phosphate-buffered saline(PBS). Formalin (final concentration, 0.1%) was added to inactivate thepurified PR8 virus at 4° C. for 1 week. Inactivation of the virus wasconfirmed by passaging viruses twice in MDCK cells and examining thecytopathic effect or lack thereof.

Experimental Infection of Mice with PB2-KO Virus.

To test the safety of the PB2-KO virus in mice, six 4-week-old femaleBALB/c mice were intranasally inoculated with 10⁶ PFU/mouse of thevirus. The body weight and survival of the infected mice were monitoreddaily for 14 days postinoculation. Also, on days 1, 3, and 6postinoculation, lungs and nasal turbinates from the inoculated micewere harvested, homogenized, and subjected to plaque assays to detectthe presence of virus.

Immunization and Protection Test.

Eight-, six-, or four-week-old female BALB/c mice (3 mice per group)were anesthetized with isoflurane and intranasally inoculated with 50 μlof medium (MEM-containing 0.3% bovine serum albumin fraction V),formalin-inactivated PR8 (64 hemagglutination units, which is equivalentto 10⁶ PFU of the PB2-KO virus), or PB2-KO virus (10⁶ PFU) once, twice,or three times at 2-week intervals, respectively. Three weeks after thefinal inoculation, mice were intranasally challenged with 0.5 or 5 50%mouse lethal doses (MLD₅₀) of PR8 virus. On days 3 and 6 postinfection,lungs and nasal turbinates of mice (3 mice per group) were collected,homogenized in 1 ml of PBS by using TissueLyser II (Qiagen, Valencia,Calif.), and clarified by low-speed centrifugation (5,000 rpm for 10minutes at 4° C.). Virus titers in homogenates were determined by usingplaque assays with AX4/PB2 cells. The body weight and survival of theremaining challenged mice (3 mice per group) were monitored daily for 14days.

Detection of Virus-Specific Antibodies.

Sera from mice (3 mice per group) were obtained via mandibular veinbleeding prior to each immunization and via the femoral artery 1 daybefore challenge. Nasal wash and bronchoalveolar lavage (BAL) fluidsamples (3 mice per group) were also obtained 1 day before challengefrom mice sacrificed by cervical dislocation. Incisions were made toinsert a cannula into the trachea. The lungs were then perfused with 1ml of PBS by using a syringe. The lavage fluid was recovered and storedin microtubes on ice. Nasal wash was collected by passing 400 μl of PBSthrough the nasal cavity. IgG and IgA antibodies in the sera, nasalwashes, and BAL fluid samples were detected by using an enzyme-linkedimmunosorbent assay (ELISA) as previously described (Kida et al., 1982).Each well was coated with purified PR8 disrupted with 0.05 M Tris-HCl(pH 7.8) containing 0.5% Triton X-100 and 0.6 M KCl. After incubation ofthe virus-coated plates with the test samples, IgA and IgG antibodies inthe samples were detected by use of goat anti-mouse IgA or IgGantibodies conjugated to horseradish peroxidase (Kirkegaard & PerryLaboratories, Inc., Gaithersburg, Md.). Neutralizing antibody titers insera of immunized mice were also evaluated as previously described(Iwatsuki-Horimoto et al., 2011). Briefly, virus (100 50% tissue cultureinfectious doses [TCID₅₀]) was incubated with 2-fold serial dilutions ofreceptor-destroying enzyme-treated sera for 30 minutes at 33° C., andthe mixtures were added to confluent MDCKcells on 96-well microplates todetermine the neutralizing activity.

IFA for Detection of Antibodies Against GFP.

293 cells grown in 35-mm glass-bottom dishes (Asahi Techno Glass) weretransfected with a plasmid expressing GFP and incubated for 48 hoursprior to the immunofluorescence assay (IFA). Cells were fixed in PBScontaining 4% paraformaldehyde (Wako Pure Chemical Industries Ltd.) for15 min and permeabilized with 0.1% Triton X-100 for 5 minutes. They wereincubated for 1 hour with 20-fold-diluted serum collected from mice mockimmunized with medium or immunized with formalin-inactivated PR8 or withthe PB2-KO virus. Anti-GFP antibody (clone GFP-20;Sigma-Aldrich)-treated cells served as a positive control. All cellswere then further incubated for 1 hour with an Alexa Fluor 594-labeledgoat anti-mouse secondary antibody (Invitrogen) and Hoechst 33342(Invitrogen) for the detection of GFP antibody and nuclear staining,respectively. Samples were observed under a confocal laser microscope(LSM510META; Carl Zeiss, Jena, Germany).

Results

Characterization of the PB2-KOvirus in Mice.

The PB2-KO virus was replication incompetent in AX4 cells but yieldedhigh titers similar to those of PR8 in AX4/PB2 cells. To determinewhether the PB2-KO virus could serve as an influenza vaccine, its safetyprofile was assessed in mice by intranasally inoculating each mouse withthe PB2-KO virus (10⁶ PFU in 50 μL) and monitoring body weight for 2weeks. Mice steadily achieved stable growth increments and appearedunperturbed by PB2-KO virus infection (data not shown). Lungs and nasalturbinates obtained on days 1, 3, and 6 postinoculation were homogenizedand subjected to plaque assays in AX4/PB2 cells to assess the growth ofthe PB2-KO virus in mice. No plaques were detected from organs of miceinfected with the PB2-KO virus, whereas a high virus titer (10⁸ PFU/g)was found in lung tissue of mice infected with 10⁶ PFU of PR8 virus.These results indicate that the PB2-KO virus did not grow in mice andthat reversion to a replication-competent virus did not occur.

Virus-Specific Antibody Responses in Mice Inoculated with the PB2-KOVirus.

The level of antibody responses elicited by the PB2-KO virus wasexamined in mice that were intranasally inoculated with the PB2-KO virusonce, twice, or three times at 2-week intervals. For comparison, micewere also mock immunized with medium or immunized withformalin-inactivated PR8 virus at a dose equivalent to 10⁶ PFU of thePB2-KO virus. Sera were collected at various time points to determinethe presence of different levels of antibodies over time. Also, at 3weeks after the final inoculation, the levels of IgG and IgA antibodiesagainst PR8 in the sera, nasal washes, and BAL fluid samples wereexamined by using an ELISA (FIG. 8). Neither the IgG nor the IgAresponse in any sample was appreciable in mice inoculated with medium.In contrast, mice immunized with the formalin-inactivated PR8 and PB2-KOviruses exhibited a time-dependent increase in serum IgG and IgA levels.After three immunizations, similar antibody levels were detected in bothinactivated virus- and PB2-KO virus-immunized mice. Interestingly, whenmice were immunized once or twice, significantly higher serum IgG or IgAtiters, respectively, were observed in PB2-KO virus-immunized mice thanin mice immunized with the formalin-inactivated virus (FIG. 8, toppanel). In nasal washes of mice inoculated with PB2-KO virus twice andin BAL fluids of mice inoculated once and twice, IgG and IgA levels weresignificantly higher than those in mice inoculated with theformalin-inactivated virus (FIG. 8, middle and bottom panels,respectively). Thus, PB2-KO virus efficiently induced IgG and IgAantibody responses in this murine model. Sera obtained from mice mockimmunized with medium had no neutralizing antibodies, whereas those fromPB2-KO-treated mice had neutralizing antibody titers of 1:16, which wasapproximately 2-fold higher than that in mice treated with inactivatedvirus (data not shown). Neutralizing activities were not detected in anynasal wash sample or BAL fluid (data not shown).

Vaccine Efficacy of the PB2-KO Virus.

To assess the vaccine efficacy of the PB2-KO virus, mice were challengedwith 0.5 or 5 MLD₅₀ of PR8 virus. The former challenge dose was testedto mimic natural infections, in which individuals are usually infectedwith a relatively low virus dose (certainly not a lethal dose).

(i) Body Weight Changes and Survival of Immunized Mice after Challenge.

To assess the vaccine efficacy of the PB2-KO virus, body weight changesand survival of mice immunized with the PB2-KO virus were examined afterthey were challenged with the PR8 virus. Mice mock immunized with mediumand challenged with 0.5 MLD₅₀ of PR8 experienced substantial body weightloss, which they subsequently recovered (FIG. 7, left panels). On theother hand, all mice mock immunized with medium and challenged with 5MLD₅₀ of PR8 showed substantial body weight loss and died atapproximately 1 week postinfection (FIG. 2, right panels). Miceimmunized once with the formalin-inactivated or PB2-KO virus experiencedweight loss (15%) after each challenge dose (FIG. 7A). It is noteworthythat 100% of mice immunized once with the PB2-KO virus survived, whereasone out of three mice immunized once with the formalin-inactivated virusdied on day 8 postinfection after being challenged with 5 MLD₅₀ of thePR8 virus (data not shown). All mice immunized twice and three timeswith the inactivated and PB2-KO viruses survived without any appreciablebody weight loss (FIGS. 7B and C).

(ii) Virus Replication in Lungs and Nasal Turbinates.

To evaluate virus replication in the lungs and nasal turbinates of miceimmunized with the PB2-KO virus, both organs were collected on days 3and 6 post-challenge with the PR8 virus. FIG. 9 shows the extent ofvirus replication in these organs. The PR8 virus replicated to a hightiter in the lungs and nasal turbinates of all mock-immunized mice.Although the potency of the PB2-KO vaccine was similar to that of theformalin-inactivated vaccine in mice immunized once, in mice thatreceived two or three vaccinations, the PB2-KO vaccine was moreefficacious than the formalin-inactivated vaccine, with virus titers inboth organs being considerably lower in mice immunized with the formerthan in those immunized with the latter. Taken together, these resultsindicate that the PB2-KO virus has better potency as an influenzavaccine than the formalin-inactivated virus.

Detection of Antibodies Against GFP in Mice Inoculated with the PB2-KOVirus.

Finally, it was determined whether the PB2-KO virus could induceantibodies against GFP, because the PB2-KO virus used here possesses theGFP gene in its PB2-coding region and GFP was expressed in PB2-KOvirus-infected culture cells (data not shown). The detection of ananti-GFP antibody in the sera of mice inoculated with the PB2-KO viruswould suggest the potential for this system as a platform for thedevelopment of an influenza virus-based multivalent vaccine. Therefore,sera was collected from mice on day 3 postchallenge and tested them inan IFA. GFP was not detected with sera from mock-immunized mice or fromthose immunized with the inactivated vaccine (FIG. 10); however, serafrom mice immunized with the PB2-KO virus, as well as a commercialanti-GFP antibody (which served as a positive control), detected GFPexpression. These results indicate that an antibody against GFP wasinduced in mice immunized with the PB2-KO virus, suggesting thepotential application of the PB2-KO virus as a multivalent vaccine.

Discussion

Here, it was demonstrated that a replication-incompetent PB2-KO viruselicits virus-specific protective antibody responses and that this virusalso induces antibodies against the reporter protein encoded in thecoding region of its PB2 segment. In particular, the PB2-KO vaccineprotected mice from lethal challenge with H1N1 PR8 virus, suggesting thepotential of this vaccine against influenza A infection. The ability todetect antibody against GFP in sera of mice inoculated with PB2-KO virussuggested that if the reporter gene, or GFP in this case, were to bereplaced with the antigenic region of another pathogen, mice inoculatedwith the recombinant virus will express antibodies against thissecondary pathogen; in turn suggesting its potential as a multivalentvaccine. Therefore, replication-incompetent PB2-KO virus can serve as aplatform for an influenza vaccine as well as for a multivalent vaccineif the PB2-coding region is replaced with the antigenic portion ofanother pathogen.

Inactivated and live-attenuated vaccines including gene knock-outviruses have been reported to successfully immunize mice against lethalinfluenza infections. The M2-KO virus lacking transmembrane andcytoplasmic tail domains efficiently replicates in M2-expression cellsand demonstrates potential as a live attenuated vaccine (Watanabe etal., 2009). The HA-KO was also shown to undergo multiple replicationcycles in cells that constitutively express the HA protein(Martinez-Sobrido et al., 2010); however, large quantities of virusesyielding sufficient HA protein in high titers seem to be required forprotective efficacy. The stable incorporation and maintenance of thereporter gene has not been studied in the M2-KO or HA-KO systems.Previously, it was demonstrated that replication-incompetent virus-likeparticles (VLPs) efficiently elicit mucosal and systemic immuneresponses in a murine model. VLPs that lack NS2 protect mice againstvarious lethal doses of influenza viruses (Watanabe et al., 2002).However, the absence of a cell line that constitutively expresses NS2precludes the efficient production of sufficient VLPs to elicitprotective efficacy.

In contrast to the M2-KO, HA-KO and/or VLPs described above, PB2-KOvirus could be prepared in a cell line that expressed PB2, yielded hightiters, and stably incorporated and maintained a GFP gene during virusreplication (Ozawa et al., 2011). These data clearly establish thefeasibility of using this system for efficient vaccine production

Safety is of utmost importance when the potential use of viruses asvaccines is concerned. Both live attenuated and most inactivatedinfluenza vaccines are currently propagated in embryonated chicken eggs,although cell-based vaccines have been licensed in Europe. Since aprerequisite for successful egg-based vaccine propagation is theselection of variants adapted to embryonated chicken eggs at the time ofimplementation, the virus in the vaccine may be slightly different fromthe circulating viruses in terms of antigenicity (Fulvini et al., 2011;Hardy et al., 1995; Robertson, 1993). Because of the propensity of eggproteins in these vaccines to induce allergies, parenterallyadministered inactivated vaccines produced in eggs are associated withadverse or anaphylactic reactions in some individuals (Halperin et al.,2002). An added complication is the possible depletion of chicken stocksin the event of an outbreak of a highly pathogenic avian influenzapandemic, which could compromise mass vaccine production (Hampson,2008).

In contrast, cell-based alternatives offer several advantages overconventional egg-based vaccine propagation. Manufacturing capacity canbe readily scaled up in proportion to demand. In addition, unlike forviruses grown in eggs, the antigenicity of viruses grown in cellsmatches that in animals and humans (Katz et al., 1990; Robertson et al.,1991).

A cell line was established that stably expresses PB2 and PB2-KO virusefficiently replicated in this cell line (i.e., at a level comparable tothat for wild-type virus) (Ozawa et al., 2011). In cells that do notexpress PB2 in trans, replication-incompetent PB2-KO virus onlyundergoes a single cycle of replication and will not result in theformation of infectious particles; thus PB2-KO virus induces aprotective antibody response without allowing the replication ofinfectious virus. Therefore, a cell-based PB2-KO vaccine eliminatesvarious obstacles to vaccine preparation and delivery.

Furthermore, knocking out the PB2 gene renders the PR8 influenza virusreplication incompetent with no evidence of recombination between therecombinant PB2 vRNA and the PB2 protein mRNA even upon multiplereplication cycles.

A formalin-inactivated vaccine efficiently protected mice fromchallenges with lethal doses of the PR8 virus by eliciting immuneresponses (FIGS. 7-9). However, even though the outcomes in terms ofsurvival and body weight loss were similar for mice immunized with theformalin-inactivated vaccine and those immunized with the PB2-KO vaccine(FIG. 7), the virus titers in the lungs and nasal turbinates of the miceimmunized with the former vaccine were higher than in those in miceimmunized with the latter (FIG. 9). This finding likely reflectsdifferences in the levels of immune responses (FIG. 8). It is alsoplausible that cytotoxic T lymphocyte (CTL) responses were activated bythe PB2-KO virus but not by the formalin-inactivated virus, sinceinactivated antigens are thought not to induce CTL responses, althoughCTL responses were not examined in this study.

By definition, a multi-valent or poly-valent vaccine refers to a vaccinedesigned to elicit an immune response to more than one infectious agentor to several different antigenic determinants of a single agent. Basedon the fact that other reporter genes and the HA and NA genes ofdifferent virus strains can be accommodated by the PB2-KO virus, thedesign and manufacturing of a multi-valent vaccine is made feasible. Asa result, it is conceivable that the PB2-KO vaccine may conferprotection against several different antigenic strains of influenza, orsubtypes of influenza and/or other pathogens. An added advantageincludes the possibility of mucosal delivery of vaccine precluding theuse of needles for subcutaneous injection of vaccine and so forth.

In conclusion, given that the PB2-KO virus elicited effective immuneresponses, induced antibodies against the product of a reporter geneencoded in its PB2 segment, is easily propagated, and can be safelyadministered as a vaccine, the PB2-KO virus represents a credible, safe,and efficacious vaccine candidate.

Example IV

Streptococcus pneumoniae is a respiratory pathogen that causes secondarybacterial infection following influenza virus infection, which isassociated with elevated mortality in the elderly. Parainfluenzaviruses, such as respiratory syncytial virus and human parainfluenzavirus type 1, are respiratory pathogens that cause severe manifestationsin infants. No vaccines are currently available for parainfluenza virus.The PB2-KO virus could be used as a multivalent vaccine because anantibody against the reporter gene product (GFP in this case) encoded inthe coding region of the PB2 segment was induced in place of authenticPB2 (FIG. 10). If major antigens of pathogens are similarly encoded inthe coding region of the PB2 segment, the PB2-KO virus could induceimmune responses against those antigens as well as against influenzaviral proteins, thereby protecting infants and the elderly from theseserious respiratory diseases.

FIG. 11 shows expression of PspA of Streptococcus pneumonia andinfluenza virus antigen in cells having PB2-KO-PspA, and expression ofinfluenza virus antigen in cells having PB2-KO-GFP. PB2-KO-PspA hasgrowth kinetics similar to wild-type influenza virus in cells thatexpress PB2 in trans but is unable to expand in cells that do notexpress PB2 in trans (FIG. 12).

Mice infected with PB2-KO-PspA or PB2-KO-GFP have influenza specific IgGand IgA in sera, BAL and nasal washes (FIG. 13), and mice infected withPB2-KO-PspA, but not PB2-KO-GFP, have pneumococcal specific IgG in sera(FIG. 14), and BAL and nasal washes (FIG. 15).

Mice immunized three times with PB2-KO-PspA survived challenge withinfluenza virus (FIG. 16) and Streptococcus pneumonia (FIG. 19).Influenza virus in the control and immunized mice could be detected innasal turbinates and lung at day 3 post-challenge (FIG. 17).Post-challenge, bacterial load was reduced in PB2-KO-PspA, but notPB2-KO-GFP, immunized mice (FIG. 18).

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification, thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

What is claimed is:
 1. An isolated attenuated influenza viruscomprising: i) 8 different gene segments including a PA viral genesegment, a PB1 viral gene segment, a mutant PB2 viral gene segment, a HAviral gene segment, a NA viral gene segment, a NP viral gene segment, aM (M1 and M2) viral gene segment, and a NS (NS1 and NS2) viral genesegment, ii) 8 different gene segments including a PA viral genesegment, a PB1 viral gene segment, a mutant PB2 viral gene segment, a HAviral gene segment, a NA (NA and NB) viral gene segment, a NP viral genesegment, a M (M1 and BM2) viral gene segment and a NS (NS1 and NS2)viral gene segment, or iii) 7 different gene segments including a PAviral gene segment, a PB1 viral gene segment, a mutant PB2 viral genesegment, a HEF viral gene segment, a NP viral gene segment, a M (M1 andCM2) viral gene segment, and a NS (NS1 and NS2) viral gene segment;wherein the mutant PB2 viral gene segment includes 5′ and 3′incorporation sequences including 3′ or 5′ coding and non-codingincorporation sequences flanking a heterologous nucleotide sequence anddoes not include contiguous sequences corresponding to sequencesencoding a functional PB2.
 2. The virus of claim 1 wherein theheterologous nucleotide sequence comprises reporter gene sequences. 3.The virus of claim 1 wherein the heterologous nucleotide sequencecomprises sequences for an antigen.
 4. The virus of claim 3 wherein theantigen is a glycoprotein.
 5. The virus of claim 1 which comprises H1,H2, H3, H5, H7, or H9 HA.
 6. A method to immunize a vertebrate,comprising: contacting the vertebrate with the virus of claim
 1. 7. Themethod of claim 6 wherein the vertebrate is an avian or a mammal.
 8. Ahost cell comprising a vector expressing influenza virus PB2 whichvector is stably integrated into the host cell genome.
 9. The host cellof claim 9 further comprising one or more vectors which includetranscription cassettes for vRNA production and transcription cassettesfor mRNA production, wherein the transcription cassettes for vRNAproduction are a transcription cassette comprising a PolI promoteroperably linked to an influenza virus PA DNA in an orientation for vRNAproduction linked to a PolI transcription termination sequence, atranscription cassette comprising a PolI promoter operably linked to aninfluenza virus PB1 DNA in an orientation for vRNA production linked toa PolI transcription termination sequence, a transcription cassettecomprising a PolI promoter operably linked to a mutant influenza virusPB2 DNA in an orientation for vRNA production linked to a PolItranscription termination sequence, a transcription cassette comprisinga PolI promoter operably linked to an influenza virus HA DNA in anorientation for vRNA production linked to a PolI transcriptiontermination sequence, a transcription cassette comprising a PolIpromoter operably linked to an influenza virus NA DNA in an orientationfor vRNA production linked to a PolI transcription termination sequence,a transcription cassette comprising a PolI promoter operably linked toan influenza virus NP DNA in an orientation for vRNA production linkedto a PolI transcription termination sequence, a transcription cassettecomprising a PolI promoter operably linked to an influenza virus M DNAin an orientation for vRNA production linked to a PolI transcriptiontermination sequence, and a transcription cassette comprising a PolIpromoter operably linked to an influenza virus NS (NS1 and NS2) DNA inan orientation for vRNA production linked to a PolI transcriptiontermination sequence, wherein the mutant PB2 DNA includes 5′ and 3′incorporation sequences including 3′ or 5′ coding and non-codingincorporation sequences flanking a heterologous nucleotide sequence anddoes not include contiguous sequences corresponding to sequences thatencode a functional PB2; and wherein the transcription cassettes formRNA production are a transcription cassette comprising a PolII promoteroperably linked to a DNA coding region for influenza virus PA linked toa PolII transcription termination sequence, a transcription cassettecomprising a PolII promoter operably linked to a DNA coding region forinfluenza virus PB1 linked to a PolII transcription terminationsequence, and a transcription cassette comprising a PolII promoteroperably linked to a DNA coding region for influenza virus NP linked toa PolII transcription termination, wherein the host cell does notcomprise sequences corresponding to PB2 coding sequences for vRNAproduction of a wild-type PB2 gene segment.
 10. The host cell of claim 9which is a 293 cell, a 293T cells, a DF-1 cell, a A549 cell, a Vero cellor a MDCK cell.
 11. A method to prepare a biologically contained 8segment influenza A or B virus, comprising contacting a host cell withone or more vectors which include transcription cassettes for vRNAproduction and transcription cassettes for mRNA production, wherein thetranscription cassettes for vRNA production are a transcription cassettecomprising a PolI promoter operably linked to an influenza virus PA DNAin an orientation for vRNA production linked to a PolI transcriptiontermination sequence, a transcription cassette comprising a PolIpromoter operably linked to an influenza virus PB1 DNA in an orientationfor vRNA production linked to a PolI transcription termination sequence,a transcription cassette comprising a PolI promoter operably linked to amutant influenza virus PB2 DNA in an orientation for vRNA productionlinked to a PolI transcription termination sequence, a transcriptioncassette comprising a PolI promoter operably linked to an influenzavirus HA DNA in an orientation for vRNA production linked to a PolItranscription termination sequence, a transcription cassette comprisinga PolI promoter operably linked to an influenza virus NA DNA in anorientation for vRNA production linked to a PolI transcriptiontermination sequence, a transcription cassette comprising a PolIpromoter operably linked to an influenza virus NP DNA in an orientationfor vRNA production linked to a PolI transcription termination sequence,a transcription cassette comprising a PolI promoter operably linked toan influenza virus M DNA in an orientation for vRNA production linked toa PolI transcription termination sequence, and a transcription cassettecomprising a PolI promoter operably linked to an influenza virus NS (NS1and NS2) DNA in an orientation for vRNA production linked to a PolItranscription termination sequence, wherein the mutant PB2 DNA includes5′ and 3′ incorporation sequences including 3′ or 5′ coding andnon-coding incorporation sequences flanking a heterologous nucleotidesequence and does not include contiguous sequences corresponding tosequences that encode a functional PB2; and wherein the transcriptioncassettes for mRNA production are a transcription cassette comprising aPolII promoter operably linked to a DNA coding region for influenzavirus PA linked to a PolII transcription termination sequence, atranscription cassette comprising a PolII promoter operably linked to aDNA coding region for influenza virus PB1 linked to a PolIItranscription termination sequence, and a transcription cassettecomprising a PolII promoter operably linked to a DNA coding region forinfluenza virus NP linked to a PolII transcription termination sequence,wherein the genome of host cell is stably augmented with a transcriptioncassette comprising a PolII promoter operably linked to a DNA codingregion for influenza virus PB2 linked to a PolII transcriptiontermination sequence, and wherein the host cell does not comprisesequences corresponding to PB2 coding sequences for vRNA production of awild-type PB2 gene segment; and isolating the biologically containedvirus from the host cell.
 12. The method of claim 11 wherein the cell iscontacted with the vector for mRNA production of PB2 before the othervectors.
 13. The method of claim 11 wherein the heterologous nucleotidesequence is a marker or selectable gene sequence.
 14. The method ofclaim 11 wherein the heterologous nucleotide sequence is a marker orselectable gene sequence.
 14. The method of claim 11 wherein thebiologically contained virus is a 6:2 reassortant.
 15. The method ofclaim 11 wherein the HA is a type A HA.
 16. The method of claim 15wherein the HA is a H1, H2, H3, H5, H7, or H9 HA.